Multispecific antibodies

ABSTRACT

The present invention relates to multispecific antibodies, methods for their production, pharmaceutical compositions containing said antibodies and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/612,104, filed Jun. 2, 2017, which is a continuation of InternationalApplication No. PCT/EP2015/078155 having an international filing date ofDec. 1, 2015, and which claims benefit under 35 U.S.C. § 119 to EuropeanPatent Application No. 14196046.8 filed Dec. 3, 2014, the entirecontents each of which are incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392041110SEQLIST.TXT,date recorded: Aug. 13, 2020, size: 134 KB).

FIELD OF THE INVENTION

The present invention relates to multispecific antibodies, methods fortheir production, pharmaceutical compositions containing said antibodiesand uses thereof.

BACKGROUND OF THE INVENTION

Engineered proteins, such as bi- or multispecific antibodies capable ofbinding two or more antigens are known in the art. Such multispecificbinding proteins can be generated using cell fusion, chemicalconjugation, or recombinant DNA techniques.

A wide variety of recombinant multispecific antibody formats have beendeveloped in the recent past, e.g. tetravalent bispecific antibodies byfusion of, e.g. an IgG antibody format and single chain domains (seee.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234).

One drawback in multispecific antibody generation is the formation ofmispaired byproducts, which have to be separated from the desiredmultispecific antibodies by sophisticated purification procedures, andreduce the production yield.

An approach to circumvent the problem of mispaired byproducts, which isknown as “knobs-into-holes technology”, aims at forcing the pairing oftwo different antibody heavy chains by introducing mutations into theCH3 domains to modify the contact interface. On one chain bulky aminoacids were replaced by amino acids with short side chains to create a“hole”. Conversely, amino acids with large side chains were introducedinto the other CH3 domain, to create a “knob”. By coexpressing these twoheavy chains (and two identical light chains, which have to beappropriate for both heavy chains), high yields of heterodimer formation(“knob-hole”) versus homodimer formation (“hole-hole” or “knob-knob”)was observed (Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; andWO 96/027011). The percentage of heterodimer could be further increasedby remodeling the interaction surfaces of the two CH3 domains using aphage display approach and the introduction of a disulfide bridge tostabilize the heterodimers (Merchant, A. M., et al., Nature Biotech. 16(1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35).

WO 2010/115598 A1 discloses trivalent bispecific antibodies based on amonospecific full length IgG molecule, wherein at the respectiveC-termini of each one of the heavy chains a variable heavy chain domainand a variable light chain domain is fused in order to form a thirdantigen binding site specifically binding to a second antigen. In orderto promote heterodimerization of the two modified heavy chains,modification of the CH3 domains according to the knobs-into-holestechnology is suggested.

Also several other antibody formats, wherein the antibody core structure(IgA, IgD, IgE, IgG or IgM) is no longer retained, have been developed;such as dia-, tria- or tetrabodies, minibodies and several single chainformats (scFv, Bis-scFv), which are capable of binding two or moreantigens (Holliger, P., et. al, Nature Biotech. 23 (2005) 1126-1136;Fischer, N., and Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et.al., J. Immunol. Methods 318 (2007) 65-74; Wu, C., et al., NatureBiotech. 25 (2007) 1290-1297). All such formats use linkers either tofuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further bindingprotein (e.g. scFv) or to fuse e.g. two Fab fragments or scFv (Fischer,N., and Léger, O., Pathobiology 74 (2007) 3-14).

WO 94/09131 discloses multispecific antibodies, wherein a first and asecond binding region formed by antibody fragments, e.g. Fab fragments,are associated with each other by associating domains that are capableof binding to each other. According to WO 94/09131 an associating domain(e.g. a VH and VL domain, respectively) is fused to each one of the Fabfragments, such that the first and second binding region are combined inorder to provide a single protein including both binding specificities.

Antibody fragments have both pros and cons as therapeutics compared withfull-size monoclonal antibodies: One advantage is that they are smallerand penetrate tissues and tumors more rapidly. In addition, the smallsize of fragments has been suggested to permit binding to epitopes notaccessible to full-sized monoclonal antibodies. On the downside,fragments demonstrate short circulating half-lives in humans, likely dueto kidney clearance. The shorter half-life may prevent sufficientaccumulation of therapy at the targeted site. Production of antibodyfragments is not trivial, as fragments are likely to form aggregates andcan be less stable than full-size monoclonal antibodies. In addition,unwanted pairing of noncongnate heavy and light chains results information of inactive antigen-binding sites and/or other non-functionalundesired side-products, which is a major problem in clinical-scaleproduction and therapeutic application of antibody fragments.

These drawbacks are overcome with the antibody format of the invention.

SUMMARY OF THE INVENTION

The present invention relates to a multispecific antibody comprising atleast three antigen binding sites, wherein two antigen binding sites areformed by a first antigen binding moiety and a second antigen bindingmoiety, wherein

-   a) a third antigen binding site is formed by a variable heavy chain    domain (VH₃) and a variable light chain domain (VL₃), wherein    -   the N-terminus of the VH₃ domain is connected to the first        antigen binding moiety via a first peptide connector, and    -   the N-terminus of the VL₃ domain is connected to the second        antigen binding moiety via a second peptide connector,-   b) the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    -   i) generation of a protuberance in one of the CH3 domains by        substituting at least one original amino acid residue by an        amino acid residue having a larger side chain volume than the        original amino acid residue, and generation of a cavity in the        other one of the CH3 domains by substituting at least one        original amino acid residue by an amino acid residue having a        smaller side chain volume than the original amino acid residue,        such that the protuberance generated in one of the CH3 domains        is positionable in the cavity generated in the other one of the        CH3 domains; or    -    substituting at least one original amino acid residue in one of        the CH3 domains by a positively charged amino acid, and        substituting at least one original amino acid residue in the        other one of the CH3 domains by a negatively charged amino acid;    -   ii) introduction of at least one cysteine residue in each CH3        domain such that a disulfide bond is formed between the CH3        domains, or    -   iii) both modifications of i) and ii);-   c) the C-terminus of the VH₃ domain of the third antigen binding    site is connected to one of the CH3 domains, and the C-terminus of    the VL₃ domain of the third antigen binding site is connected to the    other one of the CH3 domains, and-   d) the multispecific antibody is devoid of constant heavy chain    domains 2 (CH2).

In one embodiment of the invention the first antigen binding moiety is afirst Fab fragment and the second antigen binding moiety is a second Fabfragment.

One embodiment of the invention relates to a multispecific antibody,wherein

-   i) the CH3 domains are altered by generation of a protuberance in    one of the CH3 domains by substituting at least one original amino    acid residue by an amino acid residue having a larger side chain    volume than the original amino acid residue, and generation of a    cavity in the other one of the CH3 domains by substituting at least    one original amino acid residue by an amino acid residue having a    smaller side chain volume than the original amino acid residue, such    that the protuberance generated in one of the CH3 domains is    positionable in the cavity generated in the other one of the CH3    domains; or-    substituting at least one original amino acid residue in one of the    CH3 domains by a positively charged amino acid, and substituting at    least one original amino acid residue in the other one of the CH3    domains by a negatively charged amino acid;-   ii) the CH3 domains are altered by introduction of at least one    cysteine residue in each CH3 domain such that a disulfide bond is    formed between the CH3 domains; and wherein the third binding site    is disulfide stabilized by introduction of cysteine residues at the    following positions to form a disulfide bond between the VH₃ and VL₃    domains (numbering according to EU index of Kabat):    -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.

One embodiment of the invention relates to a multispecific antibody,wherein

-   i) the CH3 domains are altered by generation of a protuberance in    one of the CH3 domains by substituting at least one original amino    acid residue by an amino acid residue having a larger side chain    volume than the original amino acid residue, and generation of a    cavity in the other one of the CH3 domains by substituting at least    one original amino acid residue by an amino acid residue having a    smaller side chain volume than the original amino acid residue, such    that the protuberance generated in one of the CH3 domains is    positionable in the cavity generated in the other one of the CH3    domains; or-    substituting at least one original amino acid residue in one of the    CH3 domains by a positively charged amino acid, and substituting at    least one original amino acid residue in the other one of the CH3    domains by a negatively charged amino acid;-   ii) the CH3 domains are altered by introduction of at least one    cysteine residue in each CH3 domain such that a disulfide bond is    formed between the CH3 domains; and

wherein the third binding site is disulfide stabilized by introductionof cysteine residues in the VH₃ domain at position 44, and in the VL₃domain at position 100.

One embodiment of the invention relates to a multispecific antibody,wherein the first and second peptide connector are peptides of at least15 amino acids. One embodiment of the invention relates to amultispecific antibody, wherein the first and second peptide connectorare peptides of 15-70 amino acids. One embodiment of the inventionrelates to a multispecific antibody, wherein the first and secondpeptide connector are peptides consisting of glycine and serineresidues.

One embodiment of the invention relates to a multispecific antibody,wherein the C-terminus of the VH₃ domain is directly connected to one ofthe CH3 domains, and the C-terminus of the VL₃ domain is directlyconnected to the other one of the CH3 domains.

One embodiment of the invention relates to a trivalent multispecificantibody. One embodiment of the invention relates to a trivalent,bispecific multispecific antibody. One embodiment of the inventionrelates to a trivalent, bispecific multispecific antibody, wherein thefirst and the second antigen binding moiety specifically bind to a firstantigen, and wherein the third binding site specifically binds to asecond antigen, which is different from the first antigen.

One embodiment of the invention relates to a trivalent, trispecificmultispecific antibody.

Another aspect of the invention is a complex comprising (i) amultispecific antibody according to the invention, wherein the antibodyspecifically binds at least to a hapten and a target protein, and (ii)the hapten, which is bound by the multispecific antibody, wherein thehapten is conjugated to a therapeutic or diagnostic agent.

Another aspect of the invention is a method for the preparation of themultispecific antibody according to the invention, comprising the stepsof

-   -   transforming a host cell with expression vectors comprising        nucleic acids encoding the multispecific antibody,    -   culturing said host cell under conditions that allow synthesis        of said multispecific antibody, and    -   recovering said multispecific antibody from said host cell        culture.

Another aspect of the invention is a nucleic acid encoding themultispecific antibody according to the invention.

Another aspect of the invention is an expression vector comprising anucleic acid according to the invention.

Another aspect of the invention is a host cell comprising the expressionvector according to the invention.

Another aspect of the invention is a pharmaceutical compositioncomprising the multispecific antibody according to the invention incombination with at least one pharmaceutically acceptable carrier.

Another aspect of the invention is an immunoconjugate comprising themultispecific antibody according to the invention.

The multispecific antibodies according to the invention one the one handshow new properties due to their binding to different antigens, and onthe other hand are suitable for production and pharmaceuticalformulation due to their stability, low aggregation and pharmacokineticand biological properties (e.g. low renal clearance due to havingapproximately the same molecular weight as a full length IgG; mediumserum half-life due to avoided FcRn binding). Mispaired side productsare avoided due to use of asymmetric heterodimerization strategies. Dueto the distinctive arrangement of the at least three binding sites withrespect to each other, the antibodies according to the invention areparticularly suitable for binding to multiple antigens present on thesurface of a single cell or for binding different epitopes on oneantigen. As no CH2 domains are present in the antibodies according tothe invention, mediation of effector functions by the antibodies isabolished.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Design of multispecific antibodies according to theinvention.

FIG. 1A: Structure of a bispecific antibody according to the invention.The antibody comprising two binding arms forming a first and secondbinding site. A third antigen binding site formed by VH₃ and VL₃ domainswhich is fused to the N-terminus of respective CH3 domains. Peptideconnectors link the N-terminus of the VH₃ and VL₃ domains with the firstand second antigen binding moiety, respectively. Heterodimerization ofthe polypeptide chains including either the VH₃ or the VL₃ domain ispromoted by alteration of the CH3 domains at least one of the approachesof disulfide stabilization and CH3 engineering by the knobs-into-holestechnology (referred to in all figures as “KiH engineered”) or byintroduction of amino acids of opposite charge in correspondingpositions of the CH3/CH3 interface (referred to in all figures as“(+/−)engineered”). In addition disulfide stabilization in the VH₃/VL₃interface may be applied (not indicated).

FIG. 1B: Structure of a bispecific antibody according to the invention,wherein the first and second antigen binding moieties are Fv fragments.In this illustration, the Fv fragments bind to the same epitope.However, trispecific antibodies according to the invention may begenerated by using two Fv fragments binding to different epitopes.

FIG. 1C: Structure of a bispecific antibody according to the invention,wherein the first and second antigen binding moieties are Fab fragments.In this illustration, the Fab fragments bind to the same epitope.However, trispecific antibodies according to the invention may begenerated by using two Fab fragments binding to different epitopes.

FIGS. 2A and 2B: Design of multispecific antibodies according to theinvention comprising a first Fab fragment and a second Fab fragment.

FIG. 2A: Structure of a bispecific antibody according to the invention(right side) compared to full length IgG (left side). A third antigenbinding site formed by VH₃ and VL₃ domains, which replace the CH2domains of a full length IgG molecule. Hinge disulfides were removed toassure antigen access by connecting the Fab fragments with VH₃ and VL₃via peptide connectors lacking an interchain disulfide bond.Heterodimerization of the polypeptide chains including either the VH₃ orthe VL₃ domain is promoted by alteration of CH3 domains at least one ofthe approaches of disulfide stabilization and CH3 engineering by theknobs-into-holes technology or by introduction of amino acids ofopposite charge in corresponding positions of the CH3/CH3 interface. Inaddition disulfide stabilization in the VH₃/VL₃ interface may be applied(not indicated).

FIG. 2B: Structure of the bispecific antibodies generated in example 1.(Gly4Ser)₄ peptide connectors were used to fuse the first and second Fabfragment with either VH₃ or VL₃. In addition heterodimerization of thedifferent polypeptide chains was promoted by knobs-into-holesmodifications and disulfide stabilization in the CH3-interface as wellas disulfide stabilization of the VH₃/VL₃ binding site as outlined indetail in example 1.

FIGS. 3A-3H: Exemplary multispecific antibodies according to theinvention.

FIG. 3A: Trispecific antibody, wherein the second Fab fragment includesa domain crossover of the VH₂ and VL₂ domains, resulting in a VL₂-CH1(heavy chain), VH₂-CL (light chain) domain architecture. The first Fabfragment does not include a domain crossover, thus remaining in the wildtype domain architecture of VH₁-CH1, VL₁-CL.

FIG. 3B: Trispecific antibody, wherein the second Fab fragment includesa domain crossover of the VH-CH1 and VL-CL domains, resulting in aVL₂-CL (heavy chain), VH₂-CH1 (light chain) domain architecture. Thefirst Fab fragment does not include a domain crossover, thus remainingin the wild type domain architecture of VH₁-CH1, VL₁-CL.

FIG. 3C: Trispecific antibody, wherein the second Fab fragment includesa domain crossover of the CH1 and CL domains, resulting in a VH₂-CL(heavy chain), VL₂-CH1 (light chain) domain architecture. The first Fabfragment does not include a domain crossover, thus remaining in the wildtype domain architecture of VH₁-CH1, VL₁-CL.

FIG. 3D: Trispecific antibody, wherein the second Fab fragment includesa domain crossover of the CH1 and CL domains, resulting in a VH₂-CL(heavy chain), VL₂-CH1 (light chain) domain architecture, and the firstFab fragment includes a domain crossover, of the VH₁ and VL₁ domains,resulting in a VL₂-CH1 (heavy chain), VH₂-CL (light chain) domainarchitecture.

FIG. 3E: Trispecific antibody, wherein the second Fab fragment is asingle chain Fab fragment.

FIG. 3F: Trispecific antibody, wherein the first and the second Fabfragment are single chain Fv fragments, disulfide-stabilized Fvfragments or disulfide-stabilized single chain Fv fragments.

FIG. 3G: Trispecific antibody, wherein the first and the second Fabfragment are single domain binding sites or scaffold binding sites.

FIG. 3H: Multispecific antibody with five antigen binding sites, whereinthe first and the second Fab fragment single domain binding sites orscaffold binding sites, and wherein further single domain binding sitesor scaffold binding sites are fused to the N-terminus of the first andsecond binding site, respectively.

FIGS. 4A and 4B: Design of flexible connection between Fab fragments andthird binding site that allows antigen access.

FIG. 4A: Removal of hinge region disulfides by sufficiently long peptideconnectors without interchain disulfides allows antigen access for thirdantigen binding site. To stabilize the generated antibody format, atleast one heterodimerization approach in addition to the natural VH₃/VL₃interaction is required (indicated: disulfides in bold lines,knobs-into-holes modifications in CH3/CH3 interface).

FIG. 4B: Comparison of wild type IgG1 connection sites between CH1,hinge region and CH2 with the fusion sites of CH1, peptide connector,VH₃ domain of the generated antibody according to example 1.

FIGS. 5A and 5B: Design of stabilized interface between variable domainsof the third binding site with their respective CH3 domains.

FIG. 5A: Disulfide stabilization of both, the Fv fragment comprising VH₃and VL₃ as well as the CH3/CH3 interface leads to artificiallyintroduced cysteine residues occurring in close proximity (a) to eachother, and (b) to natural intrachain disulfide bond forming cysteines inthe respective variable or constant domains. Due to close proximity ofsaid cysteine residues potential mispairing in favor of desireddisulfide bond formation can occur leading to protein misfolding andreduction of yield (as indicated in the table).

FIG. 5B: Exemplary fusion site of VL₃ with CH3 as used in a bispecificantibody according to the invention described in example 1, wherein thethird binding site specifically binds to digoxigenin. Additionallyintroduced cysteine residues are indicated within the amino acidsequence in bold and underlined (SEQ ID NO: 01). Alternative N-terminiof CH3 domains applicable for fusion with a VH₃ or VL₃ domain areindicated below (SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04).

FIGS. 6A and 6B: Results of purification of bispecific antibodies ofexample 1.

FIG. 6A: (A) Representative chromatogram of the Kappa-select of anantibody, wherein the third binding site specifically binds todigoxigenin (BsAb CD33-Dig(SS)-CD33). (B)-(D) Size exclusionchromatography of the kappa-select binding fractions of BsAbCD33-Dig(SS)-CD33, LeY-Dig(SS)-LeY and GPC3-Dig(SS)-GPC3. Shaded boxesindicate fractions containing properly folded antibody. (E) SDS-PAGE ofpurified antibodies without (n.r.) and with (r.) sample reduction.

FIG. 6B: (A)-(C) Size exclusion chromatography of the kappa-selectbinding fractions of BsAb Dig-CD33-Dig, Dig-LeY-Dig and Dig-GPC3-Dig.Shaded boxes indicate fractions containing properly folded antibody. (D)SDS-PAGE of purified antibodies without (n.r.) and with (r.) samplereduction.

FIG. 7: Results of binding studies of bispecific antibodies ofexample 1. Simultaneous antigen binding of the antibodies generated inexample 1 as analyzed by FACS analysis on LeY-expressing MCF7 cells,CD33-expressing Molm13 cells and GPC3-expressing HepG2 cells usingDig-Cy5 as payload to address hapten binding.

FIG. 8: Results of binding studies of bispecific antibodies of example6. Simultaneous antigen binding of the BsAb BsAb LeY-Bio(SS)-LeYgenerated in example 6 as analyzed by FACS analysis on LeY-expressingand CD33 negative MCF7 cells using biotinylated Cy5 as payload toaddress hapten binding.

FIG. 9: Schematic illustration of bispecific antibody according to theinvention complexed with a toxin as payload. Indicated is a complexformed between a bispecific antibody according to the invention(complexes of antibodies with multiple specificities may be formedaccordingly) with a payload. The third binding site of a bispecificantibody according to the invention specifically binds to a hapten (e.g.digoxigenin, biotin) while the first and second binding site bind totarget molecules (e.g. tumor associated antigens, like LeY, CD33, GPC3).The complex is formed by contacting a hapten-coupled payload (e.g. atoxin, like Pseudomonas Exotoxin, PE) with the bispecific antibodyaccording to the invention. The complex may be used for targeted payloaddelivery to target-molecule expressing cells.

FIG. 10: Inhibition of MCF7 cell proliferation by a complex of BsAbLeY-Dig(SS)-LeY with digoxigenylated PE. Indicated are the results of aBrdU incorporation assay as described in detail in example 5. Y-axisindicates incorporation of BrdU into proliferating cells.

FIG. 11: Inhibition of MCF7 cell proliferation by a complex of BsAbLeY-Bio(SS)-LeY with biotinylated PE. Indicated are the results of aBrdU incorporation assay as described in detail in example 6. Y-axisindicates incorporation of BrdU into proliferating cells.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

The terms “a”, “an” and “the” generally include plural referents, unlessthe context clearly indicates otherwise.

The term “antigen binding moiety” as used herein refers to a moiety thatspecifically binds to a target antigen. The term includes antibodies aswell as other natural (e.g. receptors, ligands) or synthetic (e.g.DARPins) molecules capable of specifically binding to a target antigen.In one preferred embodiment the antigen binding moiety of an antibodyaccording to the invention is an antibody fragment.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv, scFab); and multispecific antibodies formed from antibodyfragments.

As used herein, a “Fab fragment” refers to an antibody fragmentcomprising a light chain fragment comprising a VL domain and a constantdomain of a light chain (CL), and a VH domain and a first constantdomain (CH1) of a heavy chain. As such, in case the first and secondbinding moieties are a first and second Fab fragment, respectively, thefirst Fab fragment and the second Fab fragment of such an antibodyaccording to the invention refer to two distinct Fab moieties, each onecomprising a VL and CL domain as well as a VH and CH1 domain.

An “Fv fragment” is an antibody fragment which contains a completeantigen-binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain, optionally in non-covalentassociation.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

A “recombinant antibody” is an antibody which has been produced by arecombinantly engineered host cell. It is optionally isolated orpurified.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially the entire FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

“Specificity” refers to selective recognition of a particular epitope ofan antigen by the antigen binding moiety, e.g. an antibody. Naturalantibodies, for example, are monospecific. The term “monospecificantibody” as used herein denotes an antibody that has one or morebinding sites each of which bind to the same epitope of the sameantigen. “Multispecific antibodies” bind two or more different epitopes(for example, two, three, four, or more different epitopes). Theepitopes may be on the same or different antigens. An example of amultispecific antibody is a “bispecific antibody” which binds twodifferent epitopes. When an antibody possesses more than onespecificity, the recognized epitopes may be associated with a singleantigen or with more than one antigen.

An epitope is a region of an antigen that is bound by an antibody orantigen binding moiety. The term “epitope” includes any polypeptidedeterminant capable of specific binding to an antibody or antigenbinding moiety. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,glycan side chains, phosphoryl, or sulfonyl, and, in certainembodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics.

As used herein, the terms “binding” and “specific binding” refer to thebinding of the antibody or antigen binding moiety to an epitope of theantigen in an in vitro assay, preferably in a plasmon resonance assay(BIAcore®, GE-Healthcare Uppsala, Sweden) with purified wild-typeantigen. In certain embodiments, an antibody or antigen binding moietyis said to specifically bind an antigen when it preferentiallyrecognizes its target antigen in a complex mixture of proteins and/ormacromolecules.

The affinity of the binding of an antibody to an antigen is defined bythe terms k_(a) (rate constant for the association of the antibody fromthe antibody/antigen complex), k_(D) (dissociation constant), and K_(D)(k_(D)/ka). In one embodiment binding or that/which specifically bindsto means a binding affinity (K_(D)) of 10⁻⁸ mol/l or less, in oneembodiment 10⁻⁸ M to 10⁻¹³ mol/l. Thus, an multispecific antibodyaccording to the invention specifically binds to each antigen for whichit is specific with a binding affinity (K_(D)) of 10⁻⁸ mol/l or less,e.g. with a binding affinity (K_(D)) of 10⁻⁸ to 10⁻¹³ mol/l. in oneembodiment with a binding affinity (K_(D)) of 10⁻⁹ to 10⁻¹³ mol/l.

The terms “binding site” or “antigen-binding site” as used hereindenotes the region(s) of an antigen binding molecule (e.g. an antibody)to which a ligand (e.g. the antigen or antigen fragment of it) actuallybinds and which is, preferentially, derived from an antibody. In case ofantibodies, the antigen-binding site includes antibody heavy chainvariable domains (VH) and/or antibody light chain variable domains (VL),or pairs of VH/VL. The third antigen binding site in the antibodyaccording the invention is formed by a pair of VH/VL.

Antigen-binding sites derived from antibodies that specifically bind tothe desired antigen can be derived a) from known antibodies specificallybinding to the antigen or b) from new antibodies or antibody fragmentsobtained by de novo immunization methods using inter alia either theantigen protein or nucleic acid or fragments thereof or by phagedisplay.

When being derived from an antibody, an antigen-binding site of anantibody according to the invention can contain six complementaritydetermining regions (CDRs) which contribute in varying degrees to theaffinity of the binding site for antigen. There are three heavy chainvariable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chainvariable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR andframework regions (FRs) is determined by comparison to a compileddatabase of amino acid sequences in which those regions have beendefined according to variability among the sequences. Also includedwithin the scope of the invention are functional antigen binding sitescomprised of fewer CDRs (i.e., where binding specificity is determinedby three, four or five CDRs). For example, less than a complete set of 6CDRs may be sufficient for binding.

The term “valent” as used herein denotes the presence of a specifiednumber of binding sites in an antibody molecule. A natural antibody forexample has two binding sites and is bivalent. As such, the term“trivalent” denotes the presence of three binding sites in an antibodymolecule.

The “variable domains” or “variable region” as used herein denotes eachof the pair of light and heavy chains which is involved directly inbinding the antibody to the antigen. The variable domain of a lightchain is abbreviated as “VL” and the variable domain of a heavy chain isabbreviated as “VH”. In accordance with the aforementioned, is referredherein to the variable domains of the third binding site by using “VH₃”and “VL₃”, with the number three indicating the third binding site.

The variable domains of human light chains and heavy chains have thesame general structure. Each variable domain comprises four framework(FR) regions, the sequences of which are widely conserved. The FR areconnected by three “hypervariable regions” (or “complementaritydetermining regions”, CDRs). CDRs on each chain are separated by suchframework amino acids. Therefore, the light and heavy chains of anantibody comprise from N- to C-terminal direction the domains FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. The FR adopt a beta-sheet conformationand the CDRs may form loops connecting the beta-sheet structure. TheCDRs in each chain are held in their three-dimensional structure by theFR and form together with the CDRs from the other chain an “antigenbinding site”. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

In natural antibodies the VL and VH domains are arranged terminally atthe light chains and heavy chains, respectively, which allows access ofthe antigen, thus assuring antigen binding. Within an antibody accordingto the invention the VL₃ and VH₃ domains of the third binding site maybe arranged in between two constant domains when the antigen bindingmoieties are Fab fragments. Although being embedded by constant domains,specific binding of the third binding site was surprisingly observed.

The term “constant domains” or “constant region” as used within thecurrent application denotes the sum of the domains of an antibody otherthan the variable region. The constant region is not directly involvedin binding of an antigen, but exhibits various effector functions.

Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies are divided in the “classes”: IgA, IgD, IgE,IgG and IgM, and several of these may are further divided intosubclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofantibodies are called α, δ, ε, γ and μ, respectively. The light chainconstant regions (CL) which can be found in all five antibody classesare called κ (kappa) and λ (lambda).

The “constant domains” as used herein are from human origin, which isfrom a constant heavy chain region of a human antibody of the subclassIgG1, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambdaregion. Such constant domains and regions are well known in the state ofthe art and e.g. described by Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).

The “Hinge region” is generally defined as stretching from about Glu216,or about Cys226, to about Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). The antibody according to the invention is devoid ofhinge region disulfides.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two distinct types, called kappa (κ) and lambda (λ),based on the amino acid sequences of their constant domains. A wild typelight chain typically contains two immunoglobulin domains, usually onevariable domain (VL) that is important for binding to an antigen and aconstant domain (CL).

Several different types of “heavy chains” exist that define the class orisotype of an antibody. A wild type heavy chain contains a series ofimmunoglobulin domains, usually with one variable domain (VH) that isimportant for binding antigen and several constant domains (CH1, CH2,CH3, etc.).

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. Unless otherwise specified herein, numbering of amino acidresidues in the Fc region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation. Due to the structure of the antibody according to theinvention, in particular attributed to the lack of CH2 domains, Fcmediated effector functions by an antibody according to the inventionare abolished.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted immunoglobulins (Ig) bound ontoFc receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express Fc-gammaRIII only, whereas monocytes expressFc-gammaRI, Fc-gammaRII, and Fc-gammaRIII FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362 or 5,821,337 or 6,737,056 (Presta), may beperformed. Useful effector cells for such assays include PBMC and NKcells. Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The “CH2 domain” of a human IgG Fc region usually extends from an aminoacid residue at about position 231 to an amino acid residue at aboutposition 340. The multispecific antibody is devoid of a CH2 domain. By“devoid of a CH2 domain” is meant that the antibodies according to theinvention do not comprise a CH2 domain.

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from an amino acid residue at aboutposition 341 to an amino acid residue at about position 447 of an IgG).Within an antibody according to the invention, one respective CH3 domainis arranged at the C-terminus of the VH₃ and VL₃ domain of the thirdbinding site. The “CH3 domains” herein are variant CH3 domains, whereinthe amino acid sequence of the natural CH3 domain was subjected to atleast one distinct amino acid substitution (i.e. modification of theamino acid sequence of the CH3 domain) in order to promote dimerizationof the two CH3 domains facing each other within the multispecificantibody.

Several approaches for CH3-modifications in order to supportheterodimerization have been described, for example in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954, WO 2013/096291, which are herein included by reference.

Typically, in the heterodimerization approaches known in the art, theCH3 domain of one heavy chain and the CH3 domain of the other heavychain are both engineered in a complementary manner so that the heavychain comprising one engineered CH3 domain can no longer homodimerizewith another heavy chain of the same structure. Thereby the heavy chaincomprising one engineered CH3 domain is forced to heterodimerize withthe other heavy chain comprising the CH3 domain, which is engineered ina complementary manner.

One heterodimerization approach known in the art is the so-called“knobs-into-holes” technology, which is described in detail providingseveral examples in e.g. WO 96/027011, Ridgway, J. B., et al., ProteinEng. 9 (1996) 617-621; Merchant, A. M., et al., Nat. Biotechnol. 16(1998) 677-681; and WO 98/050431, which are herein included byreference. In the “knobs-into-holes” technology, within the interfaceformed between two CH3 domains in the tertiary structure of theantibody, particular amino acids on each CH3 domain are engineered toproduce a protuberance (“knob”) in one of the CH3 domains and a cavity(“hole”) in the other one of the CH3 domains, respectively. In thetertiary structure of the multispecific antibody the introducedprotuberance in the one CH3 domain is positionable in the introducedcavity in the other CH3 domain.

Further techniques for modifying the CH3 domains of the heavy chains ofa multispecific antibody (apart from the “knobs-into-holes” technology)to enforce heterodimerization are known in the art. These technologies,especially the ones described in WO 96/27011, WO 98/050431, EP 1870459,WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954 and WO2013/096291 are contemplated herein as alternatives to the“knob-into-hole technology” in combination with a multispecific antibodyaccording to the invention. The multispecific antibody including one ofthese modification in order to support heterodimerization is furtherreferred to herein as “CH3-engineered” multispecific antibody.

According to the approach described in EP 1870459 heterodimerization ofCH3 domains is based on the introduction of charged amino acids withopposite charges at specific amino acid positions in theCH3/CH3-domain-interface between both, the first and the second heavychain (herein further referred to as a “CH3(+/−)-engineeredmultispecific antibody”).

In one embodiment of a multispecific antibody according to the inventionthe approach described in WO2013/157953 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said CH3-engineeredmultispecific antibody according to the invention, in the CH3 domain ofone heavy chain the amino acid T at position 366 (numbering according toEU index of Kabat) is substituted by K; and in the CH3 domain of theother heavy chain the amino acid L at position 351 (numbering accordingto EU index of Kabat) is substituted by D. In another embodiment of saidCH3-engineered multispecific antibody according to the invention, in theCH3 domain of one heavy chain the amino acid T at position 366(numbering according to EU index of Kabat) is substituted by K and theamino acid L at position 351 (numbering according to EU index of Kabat)is substituted by K; and in the CH3 domain of the other heavy chain theamino acid L at position 351 (numbering according to EU index of Kabat)is substituted by D.

In another embodiment of said CH3-engineered multispecific antibodyaccording to the invention, in the CH3 domain of one heavy chain theamino acid T at position 366 (numbering according to EU index of Kabat)is substituted by K and the amino acid L at position 351 (numberingaccording to EU index of Kabat) is substituted by K; and in the CH3domain of the other heavy chain the amino acid L at position 351(numbering according to EU index of Kabat) is substituted by D.Additionally at least one of the following substitutions is comprised inthe CH3 domain of the other heavy chain: the amino acid Y at position349 (numbering according to EU index of Kabat) is substituted by E, theamino acid Y at position 349 (numbering according to EU index of Kabat)is substituted by D and the amino acid L at position 368 (numberingaccording to EU index of Kabat) is substituted by E. In one embodimentthe amino acid L at position 368 (numbering according to EU index ofKabat) is substituted by E.

In one embodiment of a multispecific antibody according to the inventionthe approach described in WO2012/058768 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said CH3-engineeredmultispecific antibody according to the invention, in the CH3 domain ofone heavy chain the amino acid L at position 351 (numbering according toEU index of Kabat) is substituted by Y and the amino acid Y at position407 (numbering according to EU index of Kabat) is substituted by A; andin the CH3 domain of the other heavy chain the amino acid T at position366 (numbering according to EU index of Kabat) is substituted by A andthe amino acid K at position 409 (numbering according to EU index ofKabat) is substituted by F. In another embodiment, in addition to theaforementioned substitutions, in the CH3 domain of the other heavy chainat least one of the amino acids at positions 411 (originally T), 399(originally D), 400 (originally S), 405 (originally F), 390 (originallyN) and 392 (originally K) is substituted. Preferred substitutions are:

-   -   substituting the amino acid T at position 411 (numbering        according to EU index of Kabat) by an amino acid selected from        N, R, Q, K, D, E and W;    -   substituting the amino acid D at position 399 (numbering        according to EU index of Kabat) by an amino acid selected from        R, W, Y, and K;    -   substituting the amino acid S at position 400 (numbering        according to EU index of Kabat) by an amino acid selected from        E, D, R and K;    -   substituting the amino acid F at position 405 (numbering        according to EU index of Kabat) by an amino acid selected from        I, M, T, S, V and W;    -   substituting the amino acid N at position 390 (numbering        according to EU index of Kabat) by an amino acid selected from        R, K and D; and    -   substituting the amino acid K at position 392 (numbering        according to EU index of Kabat) by an amino acid selected from        V, M, R, L, F and E.

In another embodiment of said CH3-engineered multispecific antibodyaccording to the invention (engineered according to WO2012/058768), inthe CH3 domain of one heavy chain the amino acid L at position 351(numbering according to EU index of Kabat) is substituted by Y and theamino acid Y at position 407 (numbering according to EU index of Kabat)is substituted by A; and in the CH3 domain of the other heavy chain theamino acid T at position 366 (numbering according to EU index of Kabat)is substituted by V and the amino acid K at position 409 (numberingaccording to EU index of Kabat) is substituted by F. In anotherembodiment of said CH3-engineered multispecific antibody according tothe invention, in the CH3 domain of one heavy chain the amino acid Y atposition 407 (numbering according to EU index of Kabat) is substitutedby A; and in the CH3 domain of the other heavy chain the amino acid T atposition 366 (numbering according to EU index of Kabat) is substitutedby A and the amino acid K at position 409 (numbering according to EUindex of Kabat) is substituted by F. In said last aforementionedembodiment, in the CH3 domain of said other heavy chain the amino acid Kat position 392 (numbering according to EU index of Kabat) issubstituted by E, the amino acid T at position 411 (numbering accordingto EU index of Kabat) is substituted by E, the amino acid D at position399 (numbering according to EU index of Kabat) is substituted by R andthe amino acid S at position 400 (numbering according to EU index ofKabat) is substituted by R.

In one embodiment of a multispecific antibody according to the inventionthe approach described in WO 2011/143545 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said CH3-engineeredmultispecific antibody according to the invention, amino acidmodifications in the CH3 domains of both heavy chains are introduced atpositions 368 and/or 409.

In one embodiment of a multispecific antibody according to the inventionthe approach described in WO 2011/090762 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. WO 2011/090762 relates to amino acidmodifications according to the “knob-into-hole” technology. In oneembodiment of said CH3(KiH)-engineered multispecific antibody accordingto the invention, in the CH3 domain of one heavy chain the amino acid Tat position 366 (numbering according to EU index of Kabat) issubstituted by W; and in the CH3 domain of the other heavy chain theamino acid Y at position 407 (numbering according to EU index of Kabat)is substituted by A. In another embodiment of said CH3(KiH)-engineeredmultispecific antibody according to the invention, in the CH3 domain ofone heavy chain the amino acid T at position 366 (numbering according toEU index of Kabat) is substituted by Y; and in the CH3 domain of theother heavy chain the amino acid Y at position 407 (numbering accordingto EU index of Kabat) is substituted by T.

In one embodiment of a multispecific antibody according to theinvention, which is of IgG2 isotype, the approach described in WO2011/090762 is used to support heterodimerization of the first heavychain and the second heavy chain of the multispecific antibody.

In one embodiment of a multispecific antibody according to theinvention, the approach described in WO 2009/089004 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said CH3-engineeredmultispecific antibody according to the invention, in the CH3 domain ofone heavy chain the amino acid K or N at position 392 (numberingaccording to EU index of Kabat) is substituted by a negatively chargedamino acid (in one preferred embodiment by E or D, in one preferredembodiment by D); and in the CH3 domain of the other heavy chain theamino acid D at position 399 the amino acid E or D at position 356 orthe amino acid E at position 357 (numberings according to EU index ofKabat) is substituted by a positively charged amino acid (in onepreferred embodiment K or R, in one preferred embodiment by K, in onepreferred embodiment the amino acids at positions 399 or 356 aresubstituted by K). In one further embodiment, in addition to theaforementioned substitutions, in the CH3 domain of the one heavy chainthe amino acid K or R at position 409 (numbering according to EU indexof Kabat) is substituted by a negatively charged amino acid (in onepreferred embodiment by E or D, in one preferred embodiment by D). Inone even further embodiment, in addition to or alternatively to theaforementioned substitutions, in the CH3 domain of the one heavy chainthe amino acid K at position 439 and/or the amino acid K at position 370(numbering according to EU index of Kabat) is substituted independentlyfrom each other by a negatively charged amino acid (in one preferredembodiment by E or D, in one preferred embodiment by D).

In one embodiment of a multispecific antibody according to theinvention, the approach described in WO 2007/147901 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody. In one embodiment of said CH3-engineeredmultispecific antibody according to the invention, in the CH3 domain ofone heavy chain the amino acid K at position 253 (numbering according toEU index of Kabat) is substituted by E, the amino acid D at position 282(numbering according to EU index of Kabat) is substituted by K and theamino acid K at position 322 (numbering according to EU index of Kabat)is substituted by D; and in the CH3 domain of the other heavy chain theamino acid D at position 239 (numbering according to EU index of Kabat)is substituted by K, the amino acid E at position 240 (numberingaccording to EU index of Kabat) is substituted by K and the amino acid Kat position 292 (numbering according to EU index of Kabat) issubstituted by D.

In one embodiment of a multispecific antibody according to theinvention, the approach described in WO 2007/110205 is used to supportheterodimerization of the first heavy chain and the second heavy chainof the multispecific antibody.

In addition or alternatively to engineering the CH3 domains by aboveidentified heterodimerization strategies, the introduction of anadditional interchain disulfide bridge stabilizes the heterodimers(Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35; Merchant, A. M., etal., Nature Biotech. 16 (1998) 677-681). This is also referred to hereinas “disulfide stabilization of the CH3 domains”.

“Fused” and “connected” with respect to polypeptides refers tocomponents that are linked by peptide bonds, either directly or via oneor more peptide linkers (“peptide connector”). The term “peptide linker”or “peptide connector” as used herein interchangeably denotes a peptideof an amino acid sequence, which is preferably of synthetic origin.Typically the peptide connectors are composed of flexible residues likeglycine and serine so that the adjacent protein domains are free to moverelative to one another. Thus, typical peptide connectors used inaccordance with the invention are glycine-serine linkers, i.e. peptideconnectors consisting of a pattern of glycine and serine residues.

A first and a second peptide connector is used to fuse the first antigenbinding moiety with the VH₃ domain and the second antigen binding moietywith the VL₃ domain. However the connection between the VH₃ domain andthe VL₃ domain with their respective CH3 domain is realized directly,i.e. by direct connection of said domains, without including peptidelinkers. Hence, the term “directly connected” as used herein withrespect to the fusion/connection of polypeptides means that theconnection site does not include a peptide linker, i.e. the amino acidsequence of the fusion polypeptide solely includes the amino acidsequences of the polypeptides that were fused to each other and isdevoid of further amino acid residues of a peptide linker. This isconducted to achieve that the three-dimensional structure of

-   (a) the fusion site of VH₃ and CH3 closely mimics both, the natural    connection site of VH and CH1 as well as the natural connection site    of CH2 and CH3 of the original “parent” antibody, of which the third    binding site is derived; and-   (b) the fusion site of VL₃ and CH3 closely mimics both, the natural    connection site of VH and CH1 as well as the natural connection site    of CH2 and CH3 of the original “parent” antibody, of which the third    binding site is derived.

The term antibodies with a “domain crossover” as referred to hereinmeans antibodies, wherein in the antibody binding arm (e.g. within theFab region) deviating from the natural domain architecture of antibodiesat least one heavy chain domain was substituted by its correspondinglight chain domain and vice versa. There are three general types ofdomain crossovers, (i) the crossover of the CH1 and the CL domain, whichleads to crossover light chains of a VL-CH1 structure and crossoverheavy chains including a VH-CL structure, (ii) the crossover of the VHand the VL domain, which leads to crossover light chains of a VH-CLstructure and crossover heavy chains including a VL-CH1 structure, and(iii) the crossover of <VL-CL> and <VH-CH1> (“Fab crossover”), whichleads to crossover light chains of a VH-CH1 structure and crossoverheavy chains including a VL-CL structure (domain structures areindicated in N-terminal to C-terminal direction). Within the terms ofthe present invention “replaced by each other” with respect tocorresponding heavy and light chains refers to the aforementioned domaincrossover strategies. As such, when CH1 and CL domains are “replaced byeach other” it is referred to the domain crossover mentioned under item(i) and the resulting heavy and light chain domain architecture.Accordingly, when VH1 and VL are “replaced by each other” it is referredto the domain crossover mentioned under item (ii); and when the CH1 andCL domains are “replaced by each other” and the VH1 and VL domains are“replaced by each other” it is referred to the domain crossovermentioned under item (iii). Bispecific antibodies including domaincrossovers are disclosed, e.g. in WO 2009/080251, WO 2009/080252, WO2009/080253, WO 2009/080254 and Schaefer, W. et al, PNAS, 108 (2011)11187-1191. When antibodies according to the invention include a domaincrossover, the domain crossover is “asymmetric”, which indicates thateither (a) only one of the first and the second Fab fragment includes adomain crossover, or (b) the first and the second Fab fragment includedifferent domain crossovers indicated under items (i) to (iii) above,but not both of the first and the second Fab fragment include the samedomain crossover.

The term “tertiary structure” of an antibody as used herein refers tothe geometric shape of the antibody according to the invention. Thetertiary structure comprises a polypeptide chain backbone comprising theantibody domains, while amino acid side chains interact and bond in anumber of ways.

The term “amino acid” as used herein denotes an organic moleculepossessing an amino moiety located at α-position to a carboxylic group.Examples of amino acids include: arginine, glycine, ornithine, lysine,histidine, glutamic acid, asparagic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophane, methionine, serine, proline. Theamino acid employed is optionally in each case the L-form. The term“positively charged” or “negatively charged” amino acid refers to theamino acid side-chain charge at pH 7.4. Amino acids may be groupedaccording to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Table—Amino Acids with Specific Properties

TABLE Amino acids with specific properties 1- Side-chain Side-chaincharge Amino Acid 3-Letter Letter polarity (pH 7.4) Alanine Ala Anonpolar neutral Arginine Arg R basic polar positive Asparagine Asn Npolar neutral Aspartic acid Asp D acidic polar negative Cysteine Cys Cnonpolar neutral Glutamic acid Glu E acidic polar negative Glutamine GlnQ polar neutral Glycine Gly G nonpolar neutral Histidine His H basicpolar positive (10%) neutral (90%) Isoleucine Ile I nonpolar neutralLeucine Leu L nonpolar neutral Lysine Lys K basic polar positiveMethionine Met M nonpolar neutral Phenylalanine Phe F nonpolar neutralProline Pro P nonpolar neutral Serine Ser S polar neutral Threonine ThrT polar neutral Tryptophan Trp W nonpolar neutral Tyrosine Tyr Y polarneutral Valine Val V nonpolar neutral

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). In particular, for variabledomains and for the light chain constant domain CL of kappa and lambdaisotype, the Kabat numbering system (see pages 647-660) of Kabat, etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991) isused and is herein referred to as “numbering according to Kabat”; forthe constant heavy chain domains (CHL Hinge, CH2 and CH3) the Kabat EUindex numbering system (see pages 661-723) is used and is hereinreferred to as “numbering according to EU index of Kabat”.

Amino acid substitutions (or mutations) within the polypeptide chains ofthe multispecific antibody are prepared by introducing appropriatenucleotide changes into the antibody DNA, or by nucleotide synthesis.Such modifications can be performed, however, only in a very limitedrange. For example, the modifications do not alter the above mentionedantibody characteristics such as the IgG isotype and antigen binding,but may further improve the yield of the recombinant production, proteinstability or facilitate the purification. In certain embodiments,antibody variants having one or more conservative amino acidsubstitutions are provided.

Antibodies according to the invention are produced by recombinant means.Methods for recombinant production of antibodies are widely known in thestate of the art and comprise protein expression in prokaryotic andeukaryotic host cells with subsequent isolation of the antibody andusually purification to a pharmaceutically acceptable purity. For theexpression of the antibodies as aforementioned in a host cell, nucleicacids encoding the respective antibody light and heavy chains areinserted into expression vectors by standard methods. Expression isperformed in appropriate prokaryotic or eukaryotic host cells, like CHOcells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells,yeast, or E. coli cells, and the antibody is recovered from the cells(supernatant or cells after lysis). General methods for recombinantproduction of antibodies are well-known in the state of the art anddescribed, for example, in the review articles of Makrides, S. C.,Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

Antibodies produced by host cells may undergo post-translationalcleavage of one or more, particularly one or two, amino acids from theC-terminus of the heavy chain. Therefore an antibody produced by a hostcell by expression of a specific nucleic acid molecule encoding afull-length heavy chain may include the full-length heavy chain, or itmay include a cleaved variant of the full-length heavy chain (alsoreferred to herein as a cleaved variant heavy chain). This may be thecase where the final two C-terminal amino acids of the heavy chain areglycine (G446) and lysine (K447, numbering according to Kabat EU index).

Compositions of the invention, such as the pharmaceutical or diagnosticcompositions described herein, comprise a population of antibodies ofthe invention. The population of antibodies may comprise antibodieshaving a full-length heavy chain and antibodies having a cleaved variantheavy chain.

The term “purified” as used herein refers to polypeptides, that areremoved from their natural environment or from a source of recombinantproduction, or otherwise isolated or separated, and are at least 60%,e.g., at least 80%, free from other components, e.g. membranes andmicrosomes, with which they are naturally associated. Purification ofantibodies (recovering the antibodies from the host cell culture) isperformed in order to eliminate cellular components or othercontaminants, e.g. other cellular nucleic acids or proteins, by standardtechniques, including alkaline/SDS treatment, CsCl banding, columnchromatography, agarose gel electrophoresis, and others well known inthe art. See Ausubel, F., et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).Different methods are well established and widespread used for proteinpurification, such as affinity chromatography with microbial proteins(e.g. with affinity media for the purification of kappa orlambda-isotype constant light chain domains, e.g. KappaSelect orLambdaSelect), ion exchange chromatography (e.g. cation exchange(carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretic methods(such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi,M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

“Polynucleotide” or “nucleic acid” as used interchangeably herein,refers to polymers of nucleotides of any length, and include DNA andRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase or by asynthetic reaction. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and their analogs. A sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may comprise modification(s) made after synthesis, suchas conjugation to a label. Other types of modifications include, forexample, “caps,” substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotides(s). Further, any of the hydroxyl groupsordinarily present in the sugars may be replaced, for example, byphosphonate groups, phosphate groups, protected by standard protectinggroups, or activated to prepare additional linkages to additionalnucleotides, or may be conjugated to solid or semi-solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping group moieties of from 1 to 20 carbon atoms. Otherhydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. The term includes vectors that functionprimarily for insertion of DNA or RNA into a cell (e.g., chromosomalintegration), replication of vectors that function primarily for thereplication of DNA or RNA, and expression vectors that function fortranscription and/or translation of the DNA or RNA. Also included arevectors that provide more than one of the functions as described.

An “expression vector” is a vector are capable of directing theexpression of nucleic acids to which they are operatively linked. Whenthe expression vector is introduced into an appropriate host cell, itcan be transcribed and translated into a polypeptide. When transforminghost cells in methods according to the invention, “expression vectors”are used; thereby the term “vector” in connection with transformation ofhost cells as described herein means “expression vector”. An “expressionsystem” usually refers to a suitable host cell comprised of anexpression vector that can function to yield a desired expressionproduct.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as a transcript) is subsequentlytranslated into a peptide, polypeptide, or protein. The transcripts andthe encoded polypeptides are individually or collectively referred to asgene products. If a nucleic acid is derived from genomic DNA, expressionin a eukaryotic cell may include splicing of the corresponding mRNA.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham and Van der Eh, Virology 52 (1978) 546ff. However, other methodsfor introducing DNA into cells such as by nuclear injection or byprotoplast fusion may also be used. If prokaryotic cells or cells whichcontain substantial cell wall constructions are used, e.g. one method oftransfection is calcium treatment using calcium chloride as described byCohen, F. N, et al., PNAS 69 (1972) 7110 et seq.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

Transient expression is described by, e.g., Durocher, Y., et al., Nucl.Acids. Res. 30 (2002) E9. Cloning of variable domains is described byOrlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837;Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; andNorderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. Apreferred transient expression system (HEK 293) is described bySchlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83and by Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe composition would be administered. A pharmaceutical composition ofthe present invention can be administered by a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. To administer an antibody according to the invention by certainroutes of administration, it may be necessary to coat the antibody with,or co-administer the antibody with, a material to prevent itsinactivation. For example, the antibody may be administered to a subjectin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions.

A pharmaceutical composition comprises an effective amount of theantibodies according to the invention. An “effective amount” of anagent, e.g., an antibody, refers to an amount effective, at dosages andfor periods of time necessary, to achieve the desired therapeutic orprophylactic result. In particular, the “effective amount” denotes anamount of an antibody of the present invention that, when administeredto a subject, (i) treats or prevents the particular disease, conditionor disorder, (ii) attenuates, ameliorates or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition or disorder described herein. The therapeuticallyeffective amount will vary depending on the antibody molecules used,disease state being treated, the severity or the disease treated, theage and relative health of the subject, the route and form ofadministration, the judgment of the attending medical or veterinarypractitioner, and other factors.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. Pharmaceutically acceptable carriers includes anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. In one preferred embodiment, thecarrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g. by injection orinfusion).

The pharmaceutical compositions according to the invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Prevention of presence of microorganismsmay be ensured both by sterilization procedures, supra, and by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol, sorbic acid, and the like. It may alsobe desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, in oneembodiment the carrier is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153,Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

2. Detailed Description of the Embodiments of the Invention

I. Multispecific Antibody

The invention relates to a multispecific antibody comprising at leastthree antigen binding sites, wherein two antigen binding sites areformed by a first antigen binding moiety and a second antigen bindingmoiety, wherein

-   a) a third antigen binding site is formed by a variable heavy chain    domain (VH₃) and a variable light chain domain (VL₃), wherein    -   the N-terminus of the VH₃ domain is connected to the first        antigen binding moiety via a first peptide connector, and    -   the N-terminus of the VL₃ domain is connected to the second        antigen binding moiety via a second peptide connector,-   b) the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    -   i) generation of a protuberance in one of the CH3 domains by        substituting at least one original amino acid residue by an        amino acid residue having a larger side chain volume than the        original amino acid residue, and generation of a cavity in the        other one of the CH3 domains by substituting at least one        original amino acid residue by an amino acid residue having a        smaller side chain volume than the original amino acid residue,        such that the protuberance generated in one of the CH3 domains        is positionable in the cavity generated in the other one of the        CH3 domains (which corresponds to supporting heterodimerization        by the knobs-into-holes technology); or    -    substituting at least one original amino acid residue in one of        the CH3 domains by a positively charged amino acid; and        substituting at least one original amino acid residue in the        other one of the CH3 domains by a negatively charged amino acid        (which corresponds to supporting heterodimerization by        introducing amino acids of opposite charges within the        corresponding CH3 domains);    -   ii) introduction of at least one cysteine residue in each CH3        domain such that a disulfide bond is formed between the CH3        domains, or    -   iii) both modifications of i) and ii);-   c) the C-terminus of the VH₃ domain of the third antigen binding    site is connected to one of the CH3 domains mentioned under b), and    the C-terminus of the VL₃ domain of the third antigen binding site    is connected to the other one of the CH3 domains mentioned under b),    and-   d) the multispecific antibody is devoid of constant heavy chain    domains 2 (CH2).

A scheme of the general structure of said multispecific antibody isdepicted in FIGS. 1A-1C. The two binding arms of the multispecificantibody according to the invention formed by the first and secondantigen binding moiety may bind to the same or different antigens. Incontrast to a wild type IgG molecule, within the multispecific antibodyaccording to the invention the CH2 domains were replaced by a thirdbinding site, which is herein referred to VH₃/VL₃. As the multispecificantibody is devoid of CH2 domains, Fc mediated effector function isabolished, which is desired for several therapeutic applications. Themultispecific antibodies are particularly suitable to bind differentepitopes on the same target antigen (e.g. different epitopes on the samebiomolecule) or different biomolecules on the same cell.

Heterodimerization

In one embodiment of the multispecific antibody, the CH3 domains arealtered according to the knobs-into-holes technology. The multispecificantibody according to this embodiment is herein also referred to as“CH3(KiH)-engineered multispecific antibody” (wherein the abbreviation“KiH” stands for the “knob-into-hole technology”). Hence, according tothis embodiment within a CH3(KiH)-engineered multispecific antibody theCH3 domains are altered to promote heterodimerization by generation of aprotuberance in one of the CH3 domains by substituting at least oneoriginal amino acid residue by an amino acid residue having a largerside chain volume than the original amino acid residue; and generationof a cavity in the other one of the CH3 domains by substituting at leastone original amino acid residue by an amino acid residue having asmaller side chain volume than the original amino acid residue, suchthat the protuberance generated in one of the CH3 domains ispositionable in the cavity generated in the other one of the CH3domains.

In other words, this embodiment relates to a CH3(KiH)-engineeredmultispecific antibody according to the invention comprising a firstheavy chain and a second heavy chain, wherein in the tertiary structureof the antibody the CH3 domain of the first heavy chain and the CH3domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain each comprise a set of amino acidsthat is located within said interface in the tertiary structure of theantibody,

-   -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of one heavy chain at least one        amino acid residue is substituted by an amino acid residue        having a larger side chain volume than the original amino acid        residue, thereby generating a protuberance within the interface,        wherein the protuberance is located in the CH3 domain of the one        heavy chain, and wherein the protuberance is positionable in a        cavity located in the CH3 domain of the other heavy chain within        the interface; and    -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of the other heavy chain at least        one amino acid residue is substituted by an amino acid residue        having a smaller side chain volume than the original amino acid        residue, thereby generating a cavity within the interface,        wherein the cavity is located in the CH3 domain of the other        heavy chain, and wherein in the cavity the protuberance within        the interface located in the CH3 domain of the one heavy chain        is positionable.

In one embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention said amino acid residue having a larger sidechain volume than the original amino acid residue is selected from R, F,Y and W.

In one embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention said amino acid residue having a smaller sidechain volume than the original amino acid residue is selected from A, S,T and V.

In one embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention said amino acid residue having a larger sidechain volume than the original amino acid residue is selected from R, F,Y and W; and said amino acid residue having a smaller side chain volumethan the original amino acid residue is selected from A, S, T and V.

In one embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises a T366W mutation, and theCH3 domain of the other heavy chain (the heavy chain comprising the“hole”) comprises T366S, L368A and Y407V mutations (numberings accordingto EU index of Kabat).

In one embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises T366W and G407Y mutations,and the CH3 domain of the other heavy chain (the heavy chain comprisingthe “hole”) comprises T366S, L368A and Y407V mutations (numberingsaccording to EU index of Kabat).

In another embodiment of said CH3(KiH)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises T366W, R409D and K370Emutations, and the CH3 domain of the other heavy chain (the heavy chaincomprising the “hole”) comprises T366S, L368A, Y407V, D399K and E357Kmutations (numberings according to EU index of Kabat).

Alternatively to or in combination with the modifications according tothe knobs-into-holes technology as defined above, the CH3 domains of themultispecific antibody according to the invention are altered to promoteheterodimerization based on other heterodimerization approaches known inthe art, preferably the ones described in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954 and WO2013/096291.

In another embodiment of the multispecific antibody, alternatively to orin combination with the modifications according to the knobs-into-holestechnology the CH3 domains are altered by the introduction of chargedamino acids with opposite charges at specific amino acid positions inthe CH3/CH3-domain-interface (e.g. as described in EP 1870459). Themultispecific antibody according to this embodiment is herein alsoreferred to as “CH3(+/−)-engineered multispecific antibody” (wherein theabbreviation “+/−” stands for the oppositely charged amino acids thatwere introduced in the respective CH3 domains). Hence, according to thisembodiment within a CH3(+/−)-engineered multispecific antibody the CH3domains are altered to promote heterodimerization by substituting atleast one original amino acid residue in one of the CH3 domains by apositively charged amino acid; and substituting at least one originalamino acid residue in the other one of the CH3 domains by a negativelycharged amino acid.

In other words, this embodiment relates to a CH3(+/−)-engineeredmultispecific antibody according to the invention comprising a firstheavy chain and a second heavy chain, wherein in the tertiary structureof the antibody the CH3 domain of the first heavy chain and the CH3domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain each comprise a set of amino acidsthat is located within said interface in the tertiary structure of theantibody, wherein from the set of amino acids that is located in theinterface in the CH3 domain of one heavy chain a first amino acid issubstituted by a positively charged amino acid and from the set of aminoacids that is located in the interface in the CH3 domain of the otherheavy chain a second amino acid is substituted by a negatively chargedamino acid.

In one embodiment of said CH3(+/−)-engineered multispecific antibodyaccording to the invention the positively charged amino acid is selectedfrom K, R and H; and the negatively charged amino acid is selected fromE or D.

In one embodiment of said CH3(+/−)-engineered multispecific antibodyaccording to the invention the positively charged amino acid is selectedfrom K and R; and the negatively charged amino acid is selected from Eor D.

In one embodiment of said CH3(+/−)-engineered multispecific antibodyaccording to the invention the positively charged amino acid is K; andthe negatively charged amino acid is E.

In one embodiment of said CH3(+/−)-engineered multispecific antibodyaccording to the invention in the CH3 domain of one heavy chain theamino acid R at position 409 (numbering according to EU index of Kabat)is substituted by D and the amino acid K at position 370 (numberingaccording to EU index of Kabat) is substituted by E; and in the CH3domain of the other heavy chain the amino acid D at position 399(numbering according to EU index of Kabat) is substituted by K and theamino acid E at position 357 (numbering according to EU index of Kabat)is substituted by K.

In another embodiment of the multispecific antibody, the CH3 domains aredisulfide stabilized. The multispecific antibody according to thisembodiment is herein also referred to as “CH3(S—S)-engineeredmultispecific antibody” (wherein the abbreviation “S—S” stands for thedisulfide stabilization). Hence, according to this embodiment within aCH3(S—S)-engineered multispecific antibody the CH3 domains are alteredto promote heterodimerization by introduction of at least one cysteineresidue in each CH3 domain such that a disulfide bond is formed betweenthe CH3 domains.

In other words, this embodiment relates to a CH3(S—S)-engineeredmultispecific antibody according to the invention comprising a firstheavy chain and a second heavy chain, wherein in the tertiary structureof the antibody the CH3 domain of the first heavy chain and the CH3domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain each comprise a set of amino acidsthat is located within said interface in the tertiary structure of theantibody, from the set of amino acids that is located in the interfacein the CH3 domain of the one heavy chain a first amino acid issubstituted by cysteine; and from the set of amino acids that is locatedin the interface in the CH3 domain of the other heavy chain a secondamino acid is substituted by cysteine, wherein the second amino acid isfacing the first amino acid within the interface; such that a disulfidebridge between the CH3 domain of the one heavy chain and the CH3 domainof the other heavy chain can be formed via the introduced cysteineresidues.

In one embodiment of the CH3(S—S)-engineered multispecific antibody theCH3 domains are disulfide stabilized by a E356C or a S354C mutation inone of the CH3 domains and a Y349C mutation in the other CH3 domain(numberings according to EU index of Kabat). In one embodiment of theCH3(S—S)-engineered multispecific antibody the CH3 domains are disulfidestabilized by a S354C mutation in one of the CH3 domains and a Y349Cmutation in the other CH3 domain (numberings according to EU index ofKabat).

In yet another preferred embodiment of the multispecific antibody, theCH3 domains are disulfide stabilized and altered according to theknobs-into-holes technology. The multispecific antibody according tothis embodiment is herein also referred to as “CH3(KSS)-engineeredmultispecific antibody” (wherein the abbreviation “K” stands for theknobs-into-holes technology and the “SS” stands for the disulfidestabilization). Hence, according to this embodiment, within aCH3(KSS)-engineered multispecific antibody the CH3 domains are alteredto promote heterodimerization by generation of a protuberance in one ofthe CH3 domains by substituting at least one original amino acid residueby an amino acid residue having a larger side chain volume than theoriginal amino acid residue; and generation of a cavity in the other oneof the CH3 domains by substituting at least one original amino acidresidue by an amino acid residue having a smaller side chain volume thanthe original amino acid residue, such that the protuberance generated inone of the CH3 domains is positionable in the cavity generated in theother one of the CH3 domains; and additional introduction of at leastone cysteine residue in each CH3 domain such that a disulfide bond isformed between the CH3 domains.

In other words, this embodiment relates to a CH3(KSS)-engineeredmultispecific antibody according to the invention comprising a firstheavy chain and a second heavy chain, wherein in the tertiary structureof the antibody the CH3 domain of the first heavy chain and the CH3domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain each comprise a set of amino acidsthat is located within said interface in the tertiary structure of theantibody,

-   -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of one heavy chain at least one        amino acid residue is substituted by an amino acid residue        having a larger side chain volume than the original amino acid        residue, thereby generating a protuberance within the interface,        wherein the protuberance is located in the CH3 domain of the one        heavy chain, and wherein the protuberance is positionable in a        cavity located in the CH3 domain of the other heavy chain within        the interface; and    -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of the other heavy chain at least        one amino acid residue is substituted by an amino acid residue        having a smaller side chain volume than the original amino acid        residue, thereby generating a cavity within the interface,        wherein the cavity is located in the CH3 domain of the other        heavy chain, and wherein in the cavity the protuberance within        the interface located in the CH3 domain of the one heavy chain        is positionable; and wherein    -   from the set of amino acids that is located in the interface in        the CH3 domain of the one heavy chain a first amino acid is        substituted by cysteine; and from the set of amino acids that is        located in the interface in the CH3 domain of the other heavy        chain a second amino acid is substituted by cysteine, wherein        the second amino acid is facing the first amino acid within the        interface; such that a disulfide bridge between the CH3 domain        of the one heavy chain and the CH3 domain of the other heavy        chain can be formed via the introduced cysteine residues.

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention the E356C or S354C mutation is introduced inthe CH3 domain of the “knob” chain and the Y349C mutations areintroduced in the CH3 domain of the “hole” chain.

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention said amino acid residue having a larger sidechain volume than the original amino acid residue is selected from R, F,Y and W.

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention said amino acid residue having a smaller sidechain volume than the original amino acid residue is selected from A, S,T and V.

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention said amino acid residue having a larger sidechain volume than the original amino acid residue is selected from R, F,Y and W; and said amino acid residue having a smaller side chain volumethan the original amino acid residue is selected from A, S, T and V.

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention said amino acid residue having a larger sidechain volume than the original amino acid residue is selected from R, F,Y and W; and said amino acid residue having a smaller side chain volumethan the original amino acid residue is selected from A, S, T and V, andthe CH3 domains are disulfide stabilized by a E356C or a S354C mutationin one of the CH3 domains (in one embodiment a S354C mutation) and aY349C mutation in the other CH3 domain (numberings according to EU indexof Kabat).

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises a T366W mutation, and theCH3 domain of the other heavy chain (the heavy chain comprising the“hole”) comprises T366S, L368A and Y407V mutations (numberings accordingto EU index of Kabat), and the CH3 domains are disulfide stabilized by aE356C or a S354C mutation in one of the CH3 domains (in one embodiment aS354C mutation) and a Y349C mutation in the other CH3 domain (numberingsaccording to EU index of Kabat).

In one embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises T366W and G407Y mutations,and the CH3 domain of the other heavy chain (the heavy chain comprisingthe “hole”) comprises T366S, L368A and Y407V mutations (numberingsaccording to EU index of Kabat), and the CH3 domains are disulfidestabilized by a E356C or a S354C mutation in one of the CH3 domains (inone embodiment a S354C mutation) and a Y349C mutation in the other CH3domain (numberings according to EU index of Kabat).

In another embodiment of said CH3(KSS)-engineered multispecific antibodyaccording to the invention, the CH3 domain of the one heavy chain (theheavy chain comprising the “knob”) comprises T366W, R409D and K370Emutations, and the CH3 domain of the other heavy chain (the heavy chaincomprising the “hole”) comprises T366S, L368A, Y407V, D399K and E357Kmutations (numberings according to EU index of Kabat), the CH3 domainsare disulfide stabilized by a E356C or a S354C mutation in one of theCH3 domains (in one embodiment a S354C mutation) and a Y349C mutation inthe other CH3 domain (numberings according to EU index of Kabat).

In yet another preferred embodiment of the multispecific antibody, theCH3 domains are disulfide stabilized and altered by the introduction ofcharged amino acids with opposite charges at specific amino acidpositions in the CH3/CH3-domain-interface. The multispecific antibodyaccording to this embodiment is herein also referred to as“CH3(+/−/SS)-engineered multispecific antibody” (wherein theabbreviation “+/−” stands for the amino acids of opposite charge and the“SS” stands for the disulfide stabilization). Hence, according to thisembodiment, within a CH3((+/−/SS)-engineered multispecific antibody theCH3 domains are altered to promote heterodimerization by substituting atleast one original amino acid residue in one of the CH3 domains by apositively charged amino acid; and substituting at least one originalamino acid residue in the other one of the CH3 domains by a negativelycharged amino acid; and additional introduction of at least one cysteineresidue in each CH3 domain such that a disulfide bond is formed betweenthe CH3 domains.

In other words, this embodiment relates to a CH3(+/−/SS)-engineeredmultispecific antibody according to the invention comprising a firstheavy chain and a second heavy chain, wherein in the tertiary structureof the antibody the CH3 domain of the first heavy chain and the CH3domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain each comprise a set of amino acidsthat is located within said interface in the tertiary structure of theantibody,

-   -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of one heavy chain a first amino        acid is substituted by a positively charged amino acid; and    -   wherein from the set of amino acids that is located in the        interface in the CH3 domain of the other heavy chain a second        amino acid is substituted by a negatively charged amino acid;        and wherein    -   from the set of amino acids that is located in the interface in        the CH3 domain of the one heavy chain a first amino acid is        substituted by cysteine; and from the set of amino acids that is        located in the interface in the CH3 domain of the other heavy        chain a second amino acid is substituted by cysteine, wherein        the second amino acid is facing the first amino acid within the        interface; such that a disulfide bridge between the CH3 domain        of the one heavy chain and the CH3 domain of the other heavy        chain can be formed via the introduced cysteine residues.

In one embodiment of the invention, the third binding site of themultispecific antibody is disulfide stabilized. Hence, the VH₃ and VL₃domains are altered by introduction of at least one cysteine residue inthe VH₃ domain and one cysteine residue in the VL₃ domain such that adisulfide bond is formed between the VH₃ and VL₃ domains. In oneembodiment of the invention, the third binding site of the multispecificantibody is disulfide stabilized by introduction of cysteine residues atthe following positions to form a disulfide bond between the VH₃ and VL₃domains (numbering according to Kabat):

-   -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.

In one preferred embodiment the third binding site is disulfidestabilized by introduction of cysteine residues in the VH₃ domain atposition 44, and in the VL₃ domain at position 100.

In one preferred embodiment of the invention, the third binding site ofthe multispecific antibody is disulfide stabilized and the CH3 domainsare disulfide stabilized. Without being bound to this theory, the atleast two disulfide bonds formed by this modification in differentdomains of the altered Fc domain of the multispecific antibody accordingto the invention replace the wild type IgG hinge disulfide interactionsand thereby support heterodimerization while allowing antigen access tothe third binding site. In one embodiment, the third binding site of aCH3(S—S)-engineered multispecific antibody according to the invention isdisulfide stabilized by introduction of cysteine residues at thefollowing positions to form a disulfide bond between the VH₃ and VL₃domains (numbering according to Kabat):

-   -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.

In one preferred embodiment of the invention, the third binding site ofa CH3(S—S)-engineered multispecific antibody according to the inventionis disulfide stabilized by introduction of cysteine residues in the VH₃domain at position 44, and in the VL₃ domain at position 100.

In another preferred embodiment of the invention, the third binding siteof a CH3(S—S)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100;and the CH3 domains are disulfide stabilized by a E356C or a S354Cmutation in one of the CH3 domains and a Y349C mutation in the other CH3domain (numberings according to EU index of Kabat).

In another preferred embodiment of the invention, the heterodimerizationis supported by knobs-into-holes modifications within the CH3 domainsand, in addition, the third binding site of the multispecific antibodyand the CH3 domains are disulfide stabilized, respectively. In amultispecific antibody according to this embodiment, theheterodimerization of the knobs-into-holes modified heavy chains isfurther supported by an artificial interchain disulfide bond, whichis—in contrast to known knobs-into-holes approaches—not located withinthe CH3 domain, but in a different domain (i.e. between the VH₃ and VL₃domains). Within the antibody according to this embodimentheterodimerization of the third binding module (comprising the VH₃-CH3and VL₃-CH3 polypeptides) is promoted by four distinct interactions: (i)the natural interaction between VH₃ and VL₃, (ii) the disulfidestabilization in the VH₃/VL₃ interface, (iii) the disulfidestabilization in the CH3/CH3 interface; and (iv) the knobs-into-holesmodifications in the CH3/CH3 interface. By this, formation ofheterodimers rather than homodimer formation is promoted and stabilityof the antibody is improved.

In one embodiment, the third binding site of a CH3(KSS)-engineeredmultispecific antibody according to the invention is disulfidestabilized by introduction of cysteine residues at the followingpositions to form a disulfide bond between the VH₃ and VL₃ domains(numbering according to Kabat):

-   -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.

In one preferred embodiment of the invention, the third binding site ofa CH3(KSS)-engineered multispecific antibody according to the inventionis disulfide stabilized by introduction of cysteine residues in the VH₃domain at position 44, and in the VL₃ domain at position 100.

In another preferred embodiment of the invention, the third binding siteof a CH3(KSS)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100;and the amino acid residue having a larger side chain volume than theoriginal amino acid residue is selected from R, F, Y and W; and theamino acid residue having a smaller side chain volume than the originalamino acid residue is selected from A, S, T and V, and the CH3 domainsare disulfide stabilized by a E356C or a S354C mutation in one of theCH3 domains (in one embodiment a S354C mutation) and a Y349C mutation inthe other CH3 domain (numberings according to EU index of Kabat).

In another preferred embodiment of the invention, the third binding siteof a CH3(KSS)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100;and the CH3 domain of one heavy chain (the heavy chain comprising the“knob”) comprises a T366W mutation, and the CH3 domain of the otherheavy chain (the heavy chain comprising the “hole”) comprises T366S,L368A and Y407V mutations (numberings according to EU index of Kabat),and the CH3 domains are disulfide stabilized by a E356C or a S354Cmutation in one of the CH3 domains (in one embodiment a S354C mutation)and a Y349C mutation in the other CH3 domain (numberings according to EUindex of Kabat).

In another preferred embodiment of the invention, the third binding siteof a CH3(KSS)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100;and the CH3 domain of the one heavy chain (the heavy chain comprisingthe “knob”) comprises T366W and G407Y mutations, and the CH3 domain ofthe other heavy chain (the heavy chain comprising the “hole”) comprisesT366S, L368A and Y407V mutations (numberings according to EU index ofKabat), and the CH3 domains are disulfide stabilized by a E356C or aS354C mutation in one of the CH3 domains (in one embodiment a S354Cmutation) and a Y349C mutation in the other CH3 domain (numberingsaccording to EU index of Kabat).

In another preferred embodiment of the invention, the third binding siteof a CH3(KSS)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100;and the CH3 domain of one heavy chain (the heavy chain comprising the“knob”) comprises T366W, R409D and K370E mutations, and the CH3 domainof the other heavy chain (the heavy chain comprising the “hole”)comprises T366S, L368A, Y407V, D399K and E357K mutations (numberingsaccording to EU index of Kabat), the CH3 domains are disulfidestabilized by a E356C or a S354C mutation in one of the CH3 domains (inone embodiment a S354C mutation) and a Y349C mutation in the other CH3domain (numberings according to EU index of Kabat).

In another preferred embodiment of the invention, the heterodimerizationis supported by the introduction of charged amino acids with oppositecharges at specific amino acid positions in the CH3/CH3-domain-interfaceand, in addition, the third binding site of the multispecific antibodyand the CH3 domains are disulfide stabilized, respectively. In amultispecific antibody according to this embodiment, theheterodimerization of the modified heavy chains is further supported byan artificial interchain disulfide bond, which is not located within theCH3 domain, but in a different domain (i.e. between the VH₃ and VL₃domains). In one embodiment, the third binding site of aCH3(+/−/SS)-engineered multispecific antibody according to the inventionis disulfide stabilized by introduction of cysteine residues at thefollowing positions to form a disulfide bond between the VH₃ and VL₃domains (numbering according to Kabat):

-   -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.

In one preferred embodiment of the invention, the third binding site ofa CH3(+/−/SS)-engineered multispecific antibody according to theinvention is disulfide stabilized by introduction of cysteine residuesin the VH₃ domain at position 44, and in the VL₃ domain at position 100.

Peptide Connector

Within a multispecific antibody according to the invention, theN-terminus of the VH₃ and VL₃ domains of the third binding site arefused to the respective antigen binding moieties via a first and secondpeptide connector, respectively. In one embodiment of the invention, nointerchain disulfide bond is formed between the first and the secondpeptide connector. In one embodiment of the invention, the first andsecond peptide connectors are identical to each other.

In one embodiment of the invention, the multispecific antibody is devoidof a hinge region. In another, alternative, embodiment of the invention,the multispecific antibody comprises a natural hinge region, which doesnot form interchain disulfides. One example is the hinge region peptidederived from an antibody of IgG4 isotype.

In one preferred embodiment of the invention, the first and secondpeptide connector are peptides of at least 15 amino acids. In anotherembodiment of the invention, the first and second peptide connector arepeptides of 15-70 amino acids. In another embodiment of the invention,the first and second peptide connector are peptides of 20-50 aminoacids. In another embodiment of the invention, the first and secondpeptide connector are peptides of 10-50 amino acids. Depending e.g. onthe type of antigen to be bound by the third binding site, shorter (oreven longer) peptide connectors may also be applicable in antibodiesaccording to the invention.

In yet another embodiment of the invention, the first and second peptideconnector are approximately of the length of the natural hinge region(which is for natural antibody molecules of IgG1 isotype about 15 aminoacids, and for IgG3 isotype about 62 amino acids). Therefore in oneembodiment, wherein the multispecific antibody is of IgG1 isotype, thepeptide connectors are peptides of 10-20 amino acids, in one preferredembodiment of 12-17 amino acids. In another one embodiment, wherein themultispecific antibody is of IgG3 isotype, the peptide connectors arepeptides of 55-70 amino acids, in one preferred embodiment of 60-65amino acids.

In one embodiment of the invention, the first and second peptideconnectors are glycine-serine linkers. In one embodiment of theinvention, the first and second peptide connectors are peptidesconsisting of glycine and serine residues. In one embodiment of theinvention, the glycine-serine linkers are of the structure

(GxS)n or (GxS)nGm

-   -   with G=glycine, S=serine, x=3 or 4, n=2, 3, 4, 5 or 6, and m=0,        1, 2 or 3.

In one embodiment, of above defined glycine-serine linkers, x=3, n=3, 4,5 or 6, and m=0, 1, 2 or 3; or x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3.In one preferred embodiment, x=4 and n=2 or 3, and m=0. In yet anotherpreferred embodiment, x=4 and n=2. In one embodiment said peptideconnector is (G₄S)₂.

In one preferred embodiment of the invention, the first and secondpeptide connectors are (G₄S)₂ peptides, and the multispecific antibodyis a CH3(KSS)-engineered multispecific antibody as defined above,wherein the third binding site is disulfide stabilized by introductionof cysteine residues in the VH₃ domain at position 44, and in the VL₃domain at position 100; and wherein in the multispecific antibody theCH3 domain of the one heavy chain (the heavy chain comprising the“knob”) comprises T366W and G407Y mutations, and the CH3 domain of theother heavy chain (the heavy chain comprising the “hole”) comprisesT366S, L368A and Y407V mutations (numberings according to EU index ofKabat), and the CH3 domains are disulfide stabilized by a E356C or aS354C mutation in one of the CH3 domains (in one embodiment a S354Cmutation) and a Y349C mutation in the other CH3 domain (numberingsaccording to EU index of Kabat).

In another preferred embodiment of the invention, the first and secondpeptide connectors are (G₄S)₂ peptides, and the multispecific antibodyis a CH3(KSS)-engineered multispecific antibody as defined above,wherein the third binding site is disulfide stabilized by introductionof cysteine residues in the VH₃ domain at position 44, and in the VL₃domain at position 100; and wherein in the multispecific antibody theCH3 domain of the one heavy chain (the heavy chain comprising the“knob”) comprises T366W, G407Y and S354C mutations, and the CH3 domainof the other heavy chain (the heavy chain comprising the “hole”)comprises T366S, L368A, Y407V and Y349C mutations (numberings accordingto EU index of Kabat).

Fusion Site of the VH₃ and VL₃ Domains with Respective CH3 Domains

Within a multispecific antibody according to the invention, therespective C-terminus of the VH₃ and VL₃ domains of the third bindingsite is fused to CH3 domains. Obtaining a protein fold similar to a wildtype IgG-Fc region can be achieved best, when the variable domains VH₃and VL₃ are directly connected to the respective CH3 domains without theaid of a peptide connector. In addition, even in case the third bindingsite and the CH3/CH3 interface are both disulfide stabilized, the directconnection of the variable domains with the respective CH3 domainadvantageously prevents mispairing of the cysteine residues, which arelocated in close proximity, when forming the desired interchaindisulfide bonds.

Hence, in one embodiment of the invention the C-terminus of the VH₃domain is directly connected to one of the CH3 domains, and theC-terminus of the VL₃ domain is directly connected to the other one ofthe CH3 domains. In one preferred embodiment of the invention theC-terminus of the VH₃ domain is directly connected to one of the CH3domains, and the C-terminus of the VL₃ domain is directly connected tothe other one of the CH3 domains, wherein the connection sites aredevoid of an additional linker peptide.

In order to provide a fusion site that structurally closely mimics thenatural transition sites between variable and constant domains of wildtype antibody molecules, the C-terminus of the variable domains (VH₃ andVL₃, respectively) and/or the N-terminus of the CH3 domains (which aredirectly connected to the respective variable domains) may includemutations by substituting distinct amino acid residues.

Hence, in one embodiment of the invention, the N-terminus of the CH3domain is modified by substituting at least one original amino acidresidue. In one embodiment of the invention, the C-terminus of the VH₃domain is modified by substituting at least one original amino acidresidue. In one embodiment of the invention, the C-terminus of the VL₃domain is modified by substituting at least one original amino acidresidue.

In one preferred embodiment, the N-terminus of each one of the CH3domains includes at least one amino acid mutation, the C-terminus of theVH₃ domain includes at least one amino acid mutation, and C-terminus ofthe VL₃ domain includes at least one amino acid mutation.

In one embodiment thereof, amino acid mutations in order to improve thetertiary structure of the fusion site to mimic the natural transitionsites between variable and constant domains of wild type antibodymolecules are performed by substituting at least one amino acid residuelocated at positions 341 to 350 of the CH3 domains (numbering accordingto EU index of Kabat). In one embodiment, at least one amino acidresidue located at positions 341 to 345 of the CH3 domains issubstituted (numbering according to EU index of Kabat). In oneembodiment of the invention, the N-terminus of the CH3 domain consistsof an amino acid sequence according to SEQ ID NO: 2. In one embodimentof the invention, the N-terminus of the CH3 domain consists of an aminoacid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4.

In another embodiment, amino acid mutations in order to improve thetertiary structure of the fusion site to mimic the natural transitionsites between variable and constant domains of wild type antibodymolecules are performed by substituting at least one amino acid residueof the ten C-terminal amino acid residues of the VH₃ domain, the VL₃domain or both, the VH₃ domain and the VL₃ domain.

In one preferred embodiment, the N-terminus of each one of the CH3domains includes at least one amino acid mutation located at positions341 to 350 of the CH3 domains (numbering according to EU index ofKabat), and the ten C-terminal amino acid residues of the VH₃ domaininclude at least one amino acid mutation, and the ten C-terminal aminoacid residues of the VL₃ domain include at least one amino acidmutation.

In another preferred embodiment, the N-terminus of each one of the CH3domains includes at least one amino acid mutation located at positions341 to 345 of the CH3 domains (numbering according to EU index ofKabat), and the five C-terminal amino acid residues of the VH₃ domaininclude at least one amino acid mutation, and the five C-terminal aminoacid residues of the VL₃ domain include at least one amino acidmutation.

Antigen Binding Moieties

In one embodiment of the invention, the antigen binding moiety is aprotein specifically binding to an antigen. In one embodiment theantigen binding moiety is selected from the group of antibodies,receptors, ligands, and DARPins capable of specifically binding to anantigen. In one embodiment the antigen binding moiety is an antibodyfragment. In one preferred embodiment the antigen binding moiety isselected from the group consisting of Fv, Fab, Fab′, Fab′-SH, F(ab′)₂,and single-chain antibody molecules (e.g. scFv, scFab). In anotherpreferred embodiment the antigen binding moiety an Fv or a Fab fragment.

In another preferred embodiment of the invention, the antigen bindingmoiety is a Fab fragment. In yet another particularly preferredembodiment of the invention, the first antigen binding moiety is a firstFab fragment and the second antigen binding moiety is a second Fabfragment.

In case the first and second binding moieties of a multispecificantibody according to the invention are Fab fragments, the multispecificantibody according to the invention has an IgG like shape and exhibits acomparable molecular weight as a wild type IgG molecule. Similar to awild type IgG molecule, such multispecific antibody according to theinvention comprises two binding arms based on Fab fragments. The bindingarms may be of a wild type Fab structure or comprise furthermodifications as known in the art (e.g. the Fab fragments may be singlechain Fabs, disulfide stabilized Fabs, disulfide stabilized single chainFabs, or domain crossover Fabs). In order to assure antigen binding ofthe third binding site the hinge region of a wild type antibody, whichnaturally includes stabilizing disulfide bonds, is replaced by peptideconnectors devoid of interchain disulfide bonds. Due to the lack of thestabilization arising from the removal of the natural hinge disulfidesinteraction, the altered Fc-like region of the multispecific antibody isstabilized by supporting CH3/CH3 heterodimerization by knobs-into-holesmodifications or introduction of oppositely charged amino acids and/oradditional interchain disulfides. In addition, correct assembly of thedesired antibody molecule (e.g. avoid chain mispairing like theformation of heavy chain homodimers) is thereby supported.

Hence, in one particularly preferred embodiment the invention relates toa multispecific antibody comprising at least three antigen bindingsites, wherein two antigen binding sites are formed by a first Fabfragment and a second Fab fragment, wherein

-   a) a third antigen binding site is formed by a variable heavy chain    domain (VH₃) and a variable light chain domain (VL₃), wherein    -   the N-terminus of the VH₃ domain is connected to the first Fab        fragment via a first peptide connector, and    -   the N-terminus of the VL₃ domain is connected to the second Fab        fragment via a second peptide connector,-   b) the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    -   i) generation of a protuberance in one of the CH3 domains by        substituting at least one original amino acid residue by an        amino acid residue having a larger side chain volume than the        original amino acid residue, and generation of a cavity in the        other one of the CH3 domains by substituting at least one        original amino acid residue by an amino acid residue having a        smaller side chain volume than the original amino acid residue,        such that the protuberance generated in one of the CH3 domains        is positionable in the cavity generated in the other one of the        CH3 domains (which corresponds to supporting heterodimerization        by the knobs-into-holes technology); or    -    substituting at least one original amino acid residue in one of        the CH3 domains by a positively charged amino acid; and        substituting at least one original amino acid residue in the        other one of the CH3 domains by a negatively charged amino acid        (which corresponds to supporting heterodimerization by        introducing amino acids of opposite charges within the        corresponding CH3 domains);    -   ii) introduction of at least one cysteine residue in each CH3        domain such that a disulfide bond is formed between the CH3        domains; or    -   iii) both modifications of i) and ii);-   c) the C-terminus of the VH₃ domain of the third antigen binding    site is connected to one of the CH3 domains, and the C-terminus of    the VL₃ domain of the third antigen binding site is connected to the    other one of the CH3 domains, and-   d) the multispecific antibody is devoid of constant heavy chain    domains 2 (CH2).

In one aspect the invention relates to a multispecific antibodycomprising at least three antigen binding sites, wherein two antigenbinding sites are formed by a first Fab fragment and a second Fabfragment, wherein

-   a) a third antigen binding site is formed by a variable heavy chain    domain (VH₃) and a variable light chain domain (VL₃), wherein    -   the N-terminus of the VH₃ domain is connected to the C-terminus        of the constant heavy chain domain (CH1) or the constant light        chain domain (CL) of the first Fab fragment via a first peptide        connector, and    -   the N-terminus of the VL₃ domain is connected to the C-terminus        of the constant heavy chain domain (CH1) or the constant light        chain domain (CL) of the second Fab fragment via a second        peptide connector,-   b) the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    -   i) generation of a protuberance in one of the CH3 domains by        substituting at least one original amino acid residue by an        amino acid residue having a larger side chain volume than the        original amino acid residue, and generation of a cavity in the        other one of the CH3 domains by substituting at least one        original amino acid residue by an amino acid residue having a        smaller side chain volume than the original amino acid residue,        such that the protuberance generated in one of the CH3 domains        is positionable in the cavity generated in the other one of the        CH3 domains (which corresponds to supporting heterodimerization        by the knobs-into-holes technology); or    -    substituting at least one original amino acid residue in one of        the CH3 domains by a positively charged amino acid; and        substituting at least one original amino acid residue in the        other one of the CH3 domains by a negatively charged amino acid        (which corresponds to supporting heterodimerization by        introducing amino acids of opposite charges within the        corresponding CH3 domains);    -   ii) introduction of at least one cysteine residue in each CH3        domain such that a disulfide bond is formed between the CH3        domains; or    -   iii) both modifications of i) and ii);-   c) the C-terminus of the VH₃ domain of the third antigen binding    site is connected to one of the CH3 domains, and the C-terminus of    the VL₃ domain of the third antigen binding site is connected to the    other one of the CH3 domains, and-   d) the multispecific antibody is devoid of constant heavy chain    domains 2 (CH2).

In one preferred embodiment, the first and the second binding site ofthe multispecific antibody according to the invention are formed by afirst and second Fab fragment, respectively. In one embodiment theconstant light chain domain of the first and/or the second Fab fragmentis of kappa isotype. In one embodiment the constant light chain domainof the first and/or second Fab fragment is of lambda isotype. In oneembodiment the constant light chain domain of the first Fab fragment isof kappa isotype and the constant light chain domain of the second Fabfragment is of lambda isotype.

In one embodiment of the invention, the first Fab fragment, the secondFab fragment or both, the first and the second Fab fragment aredisulfide stabilized. In one embodiment, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment aredisulfide stabilized by introduction of cysteine residues at thefollowing positions to form a disulfide bond between the correspondingVH and VL domains (numbering according to Kabat):

-   -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100.

In one preferred embodiment of the invention, the first Fab fragment,the second Fab fragment or both, the first and the second Fab fragmentare disulfide stabilized, respectively, by introduction of cysteineresidues in its VH domain at position 44, and in its VL domain atposition 100.

In another embodiment of the invention, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment aredisulfide stabilized. In one embodiment, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment aredisulfide stabilized by introduction of cysteine residues at thefollowing positions to form a disulfide bond between the correspondingVH and VL domains (numbering according to Kabat):

-   -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100, and

and the natural disulfide bond between the polypeptide chains of therespective Fab fragment is abolished by substituting at least one of theinterchain disulfide-bond-forming cysteine residues by another aminoacid residue.

In yet another embodiment of the invention, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment aredisulfide stabilized, respectively, by introduction of cysteine residuesin its VH domain at position 44, and in its VL domain at position 100;and the natural disulfide bond between the polypeptide chains of therespective Fab fragment is abolished by substituting at least one of theinterchain disulfide-bond-forming cysteine residues by another aminoacid residue. According to this embodiment, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment, arestabilized only by the artificial disulfide bond between VH at position44 and VL at position 100.

In one embodiment of the invention, the first Fab fragment, the secondFab fragment or both, the first and the second Fab fragment are singlechain Fab fragments (scFab), i.e. the domains of the Fab fragment arearranged on a single polypeptide chain. This embodiment is particularlyuseful, when the first and second Fab fragment bind to differentepitopes (and hence, the multispecific antibody is at leasttrispecific). By this, side product formation and light chain mispairing(i.e. pairing of a light chain with the wrong heavy chain therebyforming non-functional binding sites) during recombinant expression maybe reduced and the expression yield may be improved. In one preferredembodiment, exactly one of the Fab fragments (i.e. either the first Fabfragment or the second Fab fragment) is a single chain Fab fragment(while the other Fab fragment is not a single chain Fab fragment butrather built up of two polypeptide chains).

Therefore, in one preferred embodiment of a multispecific antibodyincluding at least one single chain Fab fragment, the multispecificantibody is at least trispecific. In another preferred embodiment of amultispecific antibody including at least one single chain Fab fragment,the multispecific antibody is trispecific. In yet another preferredembodiment of a multispecific antibody including at least one singlechain Fab fragment, the multispecific antibody is trivalent andtrispecific.

In one embodiment of the invention, the first Fab fragment, the secondFab fragment or both, the first and the second Fab fragment aredisulfide stabilized single chain Fab fragments (dsFab). In oneembodiment, the first Fab fragment, the second Fab fragment or both, thefirst and the second Fab fragment are single chain Fab fragments, whichare disulfide stabilized by introduction of cysteine residues at thefollowing positions to form a disulfide bond between the correspondingVH and VL domains (numbering according to Kabat):

-   -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100.

In one preferred embodiment of the invention, the first Fab fragment,the second Fab fragment or both, the first and the second Fab fragmentare single chain Fab fragments, which are disulfide stabilized,respectively, by introduction of cysteine residues in its VH domain atposition 44, and in its VL domain at position 100. In one preferredembodiment, exactly one of the Fab fragments (i.e. either the first Fabfragment or the second Fab fragment) is a single chain Fab fragment,which is disulfide stabilized by introduction of cysteine residues inits VH domain at position 44, and in its VL domain at position 100.

In another preferred embodiment, the first Fab fragment, the second Fabfragment or both, the first and the second Fab fragment are single chainFab fragments, which are disulfide stabilized by introduction ofcysteine residues at the following positions to form a disulfide bondbetween the corresponding VH and VL domains (numbering according toKabat):

-   -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100; and

the natural disulfide bond between the polypeptide chains of therespective single chain Fab fragment is abolished by substituting atleast one of the interchain disulfide-bond-forming cysteine residues byanother amino acid residue. In yet another preferred embodiment of theinvention, the first Fab fragment, the second Fab fragment or both, thefirst and the second Fab fragment are single chain Fab fragments, whichare disulfide stabilized, respectively, by introduction of cysteineresidues in its VH domain at position 44, and in its VL domain atposition 100, and the natural disulfide bond between the polypeptidechains of the respective single chain Fab fragment is abolished bysubstituting at least one of the interchain disulfide-bond-formingcysteine residues by another amino acid residue.

In one embodiment of the invention, the first Fab fragment, the secondFab fragment or both, the first and the second Fab fragment are alteredby a domain crossover, such that either:

-   a) only the CH1 and CL domains are replaced by each other;-   b) only the VH and VL domains are replaced by each other; or-   c) the CH1 and CL domains are replaced by each other and the VH and    VL domains are replaced by each other,

with the provision that in case both the first Fab fragment and thesecond Fab fragment are altered by a domain crossover, they are alteredby different domain crossovers. This means for example that in case bothFab fragments comprise a domain crossover, when the first Fab fragmentcomprises the domain crossover defined under a), i.e. the CH1 and CLdomains are replaced by each other, then the second Fab fragmentcomprises either the domain crossover defined under b) (i.e. replacementof corresponding VH and VL domains) or the domain crossover definedunder c) (i.e. replacement of VH-CH1 with VL-CL), but the second Fabfragment does not comprise the domain crossover defined under a) (i.e.only the CH1 and CL domains are replaced by each other).

Hence, the multispecific antibody according to this embodiment comprisesan asymmetric domain crossover with respect to the first and second Fabfragment, meaning that due to the domain crossover the light chains ofthe first Fab fragment and the second Fab fragment are no longercomposed of the same domain architecture but are rather comprised of adifferent domain architecture. Thereby, pairing of the light chain ofthe first Fab fragment with the heavy chain of the second Fab fragment(and vice versa) is avoided. This embodiment is particularly useful,when the first and second Fab fragment bind to different epitopes (andhence, the multispecific antibody is at least trispecific). By thisembodiment, side product formation and light chain mispairing (i.e.pairing of a light chain with the wrong heavy chain thereby formingnon-functional binding sites) during recombinant expression may bereduced and the expression yield of the antibody may be improved.

Therefore, in one preferred embodiment of a multispecific antibodyincluding a domain crossover in at least one of the Fab fragments, themultispecific antibody is at least trispecific. In another preferredembodiment of a multispecific antibody including a domain crossover inat least one of the Fab fragments, the multispecific antibody istrispecific. In yet another preferred embodiment of a multispecificantibody including a domain crossover in at least one of the Fabfragments, the multispecific antibody is trivalent and trispecific.

In one embodiment of the invention, only one of the Fab fragments (i.e.the first Fab fragment or the second Fab fragment but not both Fabfragments) is altered by a domain crossover such that only the CH1 andCL domains of the Fab fragment are replaced by each other.

In one embodiment of the invention, only one of the Fab fragments (i.e.the first Fab fragment or the second Fab fragment but not both Fabfragments) is altered by a domain crossover such that only the VH and VLdomains of the Fab fragment are replaced by each other.

In case Fab fragments are used as binding arms of the multispecificantibody according to the invention, the antibody exhibits an IgG-likestructure, however it comprises an additional binding site that replacesthe original CH2/CH2 interface.

Further binding sites may be fused to the N-termini or C-termini of theheavy chains or light chains of the multispecific antibody in order toprovide antibodies of higher valence. In one preferred embodiment themultispecific antibody is trivalent, thereby resembling the wild typethree-dimensional structure of an IgG molecule.

Binding to Different Epitopes

In one embodiment of the invention the multispecific antibody comprisesat least one polyepitopic binding site (i.e. is capable of binding totwo different epitopes on one biological molecule or two differentepitopes from different biological molecules, e.g. as disclosed in WO2008/027236 A2). By this, multispecific antibodies of more than threespecificities (e.g. tetraspecific antibodies) can be generated in asimilar structure and molecular weight as wild type IgG molecules. Inone embodiment of the invention, the first antigen binding moiety, thesecond antigen binding moiety or both, the first and the second antigenbinding moiety comprise a polyepitopic binding site. In anotherembodiment of the invention, the third binding site comprises apolyepitopic binding site. In yet another embodiment, the first and thesecond antigen binding moiety and the third binding site of themultispecific antibody comprise a polyepitopic binding site.

The multispecific antibody according to the invention is capable ofbinding to different epitopes. This may be achieved by combining bindingsites that specifically bind to a single antigen or, in addition, byincluding binding sites that are polyepitopic and hence, specificallybind to more than one epitope (in one preferred embodiment saidpolyepitopic binding site binds to two different epitopes). Thereby,trivalent multispecific antibodies may be produced that are capable ofbinding to a high number of different epitopes. In case the first andsecond antigen binding moieties are respective Fab fragments, themultispecific antibody advantageously maintains an IgG like shape andmolecular weight. The multispecific antibodies are particularly suitableto bind different epitopes on the same target antigen (e.g. differentepitopes on the same biomolecule) or different biomolecules on the samecell.

In one preferred embodiment, the multispecific antibody according to theinvention includes three binding sites each one binding to a singleepitope. Thereby, the multispecific antibody according to thisembodiment may be bispecific or trispecific.

In another preferred embodiment, the multispecific antibody according tothe invention includes at least one polyepitopic binding site (in onepreferred embodiment said polyepitopic binding site binds to twodifferent epitopes). In one embodiment, the multispecific antibody is atrispecific antibody, wherein the first and second antigen bindingmoieties include two identical polyepitopic binding sites (specificallybinding each to two different epitopes) and the third binding sitespecifically binds to another (third) epitope. In another embodiment,the multispecific antibody is a trispecific antibody, wherein the firstand second antigen binding moieties specifically bind to a first epitopeand the third binding site is a polyepitopic binding site specificallybinding to a second and a third epitope. Thereby, the multispecificantibody according to this embodiment is at least trispecific. Whencombing three different polyepitopic binding sites that each bind twodifferent epitopes, in one embodiment of the multispecific antibody, theantibody may be up to hexaspecific.

In one embodiment of the invention the antibody is bispecific. In oneembodiment of the invention the antibody is trivalent and bispecific. Inone embodiment of the invention the antibody is bispecific andspecifically binds two different antigens on one cell or two differentepitopes of the same antigen. In one embodiment of the invention theantibody is trivalent and bispecific, and specifically binds twodifferent antigens on one cell or two different epitopes of the sameantigen.

In one preferred embodiment of the invention the antibody is bispecific,wherein the first antigen binding moiety and the second antigen bindingmoiety specifically bind to the same epitope, and wherein the thirdbinding site specifically binds to a different epitope. In one preferredembodiment of the invention the antibody is trivalent and bispecific,wherein the first antigen binding moiety and the second antigen bindingmoiety specifically bind to the same epitope, and wherein the thirdbinding site specifically binds to a different epitope.

Within a bispecific antibody according to these embodiments comprising afirst and second antigen binding moiety in the form of Fab fragments, inone embodiment the first and second Fab fragment do not comprise adomain crossover. Hence, in one preferred embodiment, the light chainsof the first and second Fab fragment are composed of VL and CL domains(from N-terminal to C-terminal direction).

In another embodiment of the invention the antibody is bispecific,wherein the first antigen binding moiety and the third binding sitespecifically bind to the same epitope, and wherein the second antigenbinding moiety specifically binds to a different epitope. In anotherembodiment of the invention the antibody is trivalent and bispecific,wherein the first antigen binding moiety and the third binding sitespecifically bind to the same epitope, and wherein the second antigenbinding moiety specifically binds to a different epitope.

Within a bispecific antibody according to these embodiments comprising afirst and second antigen binding moiety in the form of Fab fragments, atleast one of the Fab fragments either comprises a domain crossover or isprovided in the form of a single chain Fab fragment. In one preferredembodiment, at least one of the Fab fragments comprises a domaincrossover as defined above (optionally including further domaincrossover embodiments such as introduction of charged amino acids intoat least one of the Fab fragments). Thereby, chain mispairing is avoidedand the expression yield of the multispecific antibody is improved.

In one embodiment of the invention the antibody is trispecific. In oneembodiment of the invention the antibody is trivalent and trispecific.

In one preferred embodiment of a trispecific antibody according to theinvention, each binding site binds to single epitope, wherein the firstantigen binding moiety, the second antigen binding moiety and the thirdbinding site specifically bind to a different epitope, respectively.

In another embodiment of a trispecific antibody according to theinvention, the first and second antigen binding moiety bind to the sameepitope and the third binding site binds to two different epitopes (andtherefore is polyepitopic).

In yet another embodiment of a trispecific antibody according to theinvention, the first and second antigen binding moiety are based on thesame, polyepitopic binding sites (each one binding to two differentepitopes), and the third binding site binds to a single epitope that isdifferent from the epitopes bound by the first and second antigenbinding moiety.

In all embodiments, wherein the antibody according to the inventioncomprises a first Fab fragment and a second Fab fragments as first andsecond antigen binding moiety and wherein said first Fab fragment andsaid second Fab fragment are based on different binding sites and hence,specifically bind to different epitopes, the Fab fragments arepreferably specifically designed to avoid light chain mispairing betweenthe light chains and heavy chains of the first Fab fragment and thesecond Fab fragment, respectively, by either using single chain Fabfragments (which may be further disulfide stabilized), or by usingdomain crossover strategies to achieve a different domain architecturein the light chains of the first Fab fragment and the second Fabfragment, thereby suppressing light chain mispairing.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentspecifically bind to different epitopes, the first Fab fragment, thesecond Fab fragment or both, the first and the second Fab fragment arealtered by a domain crossover, such that either:

-   a) only the CH1 and CL domains are replaced by each other;-   b) only the VH and VL domains are replaced by each other; or-   c) the CH1 and CL domains are replaced by each other and the VH and    VL domains are replaced by each other,

with the provision that in case both the first Fab fragment and thesecond Fab fragment are altered by a domain crossover, they are alteredby different domain crossovers.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentspecifically bind to different epitopes, only one of the Fab fragments(i.e. the first Fab fragment or the second Fab fragment but not both Fabfragments) is altered by a domain crossover such that only the CH1 andCL domains of the Fab fragment are replaced by each other.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentspecifically bind to different epitopes, only one of the Fab fragments(i.e. the first Fab fragment or the second Fab fragment but not both Fabfragments) is altered by a domain crossover such that only the VH and VLdomains of the Fab fragment are replaced by each other.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentbind to different epitopes the first Fab fragment, the second Fabfragment or both, the first and the second Fab fragment are single chainFab fragments (scFab).

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentbind to different epitopes, the first Fab fragment, the second Fabfragment or both, the first and the second Fab fragment are disulfidestabilized single chain Fab fragments (dsFab). In one embodiment of amultispecific antibody according to the invention, wherein the first Fabfragment and the second Fab fragment bind to different epitopes, thefirst Fab fragment, the second Fab fragment or both, the first and thesecond Fab fragment are single chain Fab fragments, which are disulfidestabilized by introduction of cysteine residues at the followingpositions to form a disulfide bond between the corresponding VH and VLdomains (numbering according to Kabat):

-   -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentbind to different epitopes, the first Fab fragment, the second Fabfragment or both, the first and the second Fab fragment are single chainFab fragments, which are disulfide stabilized, respectively, byintroduction of cysteine residues in its VH domain at position 44, andin its VL domain at position 100. In one preferred embodiment, exactlyone of the Fab fragments (i.e. either the first Fab fragment or thesecond Fab fragment) is a single chain Fab fragment, which is disulfidestabilized by introduction of cysteine residues in its VH domain atposition 44, and in its VL domain at position 100.

In one embodiment of a multispecific antibody according to theinvention, wherein the first Fab fragment and the second Fab fragmentbind to different epitopes, the first Fab fragment is a single chain Fabfragment (in one embodiment a disulfide stabilized single chain Fabfragment) and the second Fab fragment comprises a domain crossover asdefined above.

Antibody Isotypes

In one embodiment of the invention, the multispecific antibody comprisesimmunoglobulin constant regions of one or more immunoglobulin classes.Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes and,in the case of IgG and IgA, their subtypes. In one embodiment of theinvention, the multispecific antibody has a constant domain structure ofan IgG type antibody.

In one embodiment the constant domains of an antibody according to theinvention are of human IgG1 or IgG4 subclass. In one embodiment, the CH3domain is derived from a human IgG1 antibody. In one embodiment, themultispecific antibody is devoid of a CH4 domain.

In one embodiment of the invention the antibody is a monoclonalantibody. In one embodiment of the invention the antibody is a humanizedmonoclonal antibody. In one embodiment of the invention the antibody isa human monoclonal antibody.

In one embodiment of the invention the multispecific antibody is anisolated antibody.

In one embodiment, an antibody comprising a heavy chain including a CH3domain as specified herein, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment, an antibody comprising a heavy chainincluding a CH3 domain, as specified herein, comprises an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat).

Complex Including Antibody and Hapten-Coupled Agent for Targeted PayloadDelivery

Another object of the invention is a complex comprising (i) amultispecific antibody according to the invention, wherein the antibodyspecifically binds at least to a hapten and a target protein (therebyincluding at least one binding site specifically binding to the haptenand at least one binding site specifically binding to the targetprotein), and (ii) the hapten, which is bound by the multispecificantibody, wherein the hapten is conjugated to a therapeutic ordiagnostic agent. Within the complex, the hapten is bound to the bindingsite of the antibody, which specifically binds to the hapten. Thereby,the hapten conjugated to the therapeutic or diagnostic agent isnon-covalently coupled to the antibody. Within the complex the antibodymaintains its binding specificity and affinity while the therapeutic ordiagnostic agent coupled to the hapten maintains its activity as well.Complexes of a hapten-binding bispecific antibody with haptenylatedtherapeutic or diagnostic agent in general are known in the art, e.g.from WO 2011/1003557 A1. The complexes according to the invention may bedesigned and applied as described in WO 2011/1003557 A1, the contents ofwhich are fully incorporated herein by reference.

In one embodiment, the antibody present in the complex according to theinvention is bispecific. In one embodiment, the hapten is selected fromdigoxigenin, biotin, theophylline, fluorescein, DOTA, and DOTAM. In oneembodiment the target protein is a cell surface antigen or anintracellular antigen. In one embodiment the target protein is a cellsurface or an intracellular tumor-associated antigen. In one embodimentthe target protein is a cell surface tumor-associated antigen. In oneembodiment the target protein is Lewis Y. In one embodiment the targetprotein is CD33. In one embodiment the target protein is Glypican 3.

In one embodiment of the invention said multispecific antibody is usedas a payload delivery vehicle for the therapeutic or diagnostic agent.The therapeutic or diagnostic agent is conjugated with the hapten andthus coupled by the hapten-binding site of the multispecific antibodyaccording to the invention to form the complex according to theinvention. This complex is defined and stable and specifically deliversthe payload to a target cell or tissue. Since the haptenylatedtherapeutic or diagnostic agent is coupled in a non-covalent manner tothe multispecific antibody, the payload is stably bound to its deliveryvehicle during circulation but also gets efficiently released afterinternalization. The conjugation with the hapten does not affect theactivity of most therapeutic or diagnostic agents. The multispecificantibody thus does not contain an unusual covalently coupled payload andtherefore exhibits low risk of immunogenicity. Therefore this simpleconjugation procedure can be used for a great variety of payloadmolecules in combination with only one single multispecific antibody;the payload molecules being for example peptides, proteins, smallmolecules, imaging reagents and nucleic acids. Complexes of ahaptenylated diagnostic or therapeutic agent with the multispecificantibody according to the invention containing at least one haptenbinding site may confer benign biophysical behavior and improved PKparameters to the diagnostic or therapeutic agent, e.g. to diagnostic ortherapeutic proteins, peptides or small molecules. Furthermore, suchcomplexes are capable to target the delivery payload to cells whichdisplay the target protein antigen that is recognized by the at leastone further binding site of the multispecific antibody.

In one embodiment the therapeutic or diagnostic agent coupled to thehapten is selected from the group consisting of a peptide, a protein, asmall molecule, a radioactively labeled small molecule, a nucleic acidand an imaging agent.

In one embodiment the therapeutic or diagnostic agent is a peptide. Uponbinding of a haptenylated peptide to a multispecific antibody accordingto the invention, the peptide retains its full biological activity.Non-limiting examples of peptides are Mellitin, Fam5B, INF7, FallV1 andFallV2. One aspect of the invention is the use of the multispecificantibodies according to the invention for delivery of toxin-derivedpeptides to target-antigen-expressing tumor cells.

In one embodiment the therapeutic or diagnostic agent is a protein. Uponbinding of a haptenylated protein to a multispecific antibody accordingto the invention, the protein retains its full biological activity.

In one embodiment the therapeutic or diagnostic agent is a smallmolecule. In one embodiment the small molecule is a toxin or is a smallmolecule derived from a toxin. In one embodiment the small molecule isPseudomonas Exotoxin.

In one embodiment the therapeutic or diagnostic agent is a radioactivelylabelled small molecule. The haptenylated radioisotope or theradioisotope attached to the haptenylated small molecule displayseffective tissue penetration, fast clearance, and are retained only oncells covered by the complex according to the invention expressing thetarget protein antigen. This enables specific targeting and avoidssystemic nonspecific release of therapeutic radioisotopes. One aspect ofthe invention is the use of the multispecific antibodies according tothe invention for delivery of a haptenylated radioisotope or aradioisotope attached to a haptenylated small molecule to a diseasedtissue. In one embodiment, said diseased tissue is a tumor and thetarget protein is a tumor associated antigen.

In one embodiment the therapeutic or diagnostic agent is a nucleic acid.In one embodiment the nucleic acid is double stranded RNA (dsRNA).Double-stranded ribonucleic acid (dsRNA) molecules have been shown toblock gene expression in a highly conserved regulatory mechanism knownas RNA interference (RNAi). Hence, one aspect of the invention is the ofthe multispecific antibodies according to the invention for targetedgene therapy of targeted dsRNA delivery.

In one embodiment the therapeutic or diagnostic agent is an imagingagent. In one embodiment, the imaging agent is a fluorophor. The imagingagent retains its properties despite being haptenylated and complexed tothe antibody according to the invention. The haptenylated imaging agentdisplays effective tissue penetration, fast clearance, and are retainedonly on cells covered by the complex according to the inventionexpressing the target protein antigen. This enables effectivetime-resolved imaging, and assessment of tumor vascularization, orchanges within tumor vascularization. One aspect of the invention is theuse of the multispecific antibodies according to the invention forimaging of a diseased tissue. Another aspect of the invention is the useof the multispecific antibodies according to the invention of in vitroimaging, e.g. for FACS analyses. In one embodiment, said diseased tissueis a tumor and the target protein is a tumor associated antigen.

Another aspect is a method for the preparation of a complex according tothe invention, the method including the steps of

-   -   providing a multispecific antibody according to the invention,        wherein the multispecific antibody specifically binds to a        hapten and a target protein,    -   providing a hapten, which is specifically bound by the        multispecific antibody, wherein the hapten is conjugated to a        therapeutic or diagnostic agent, and    -   contacting the multispecific antibody with the hapten, which is        conjugated to the therapeutic or diagnostic agent.

Another aspect of the invention is the use of the complex according tothe invention as a medicament. Another aspect of the invention is theuse of the complex according to the invention for diagnostic purposes.

Another aspect of the invention is the use of the complex according tothe invention for delivery of a therapeutic or diagnostic agent to atarget cell or tissue. Another aspect of the invention is the use of thecomplex according to the invention for targeted cancer therapy. Anotheraspect of the invention is the use of the complex according to theinvention for targeted radiotherapy.

Another aspect of the invention is the use of the complex according tothe invention for imaging of cells or tissues.

Another aspect of the invention is a composition comprising a complexaccording to the invention comprising the multispecific antibodyaccording to the invention, which specifically binds to a hapten and atarget protein, and a hapten that is conjugated to a therapeutic ordiagnostic agent. In one embodiment, said composition is a diagnosticcomposition. In another embodiment the composition is a pharmaceuticalcomposition.

II. Recombinant Method

The multispecific antibody is prepared by recombinant methods. Thus, theinvention also relates to a method for the preparation of amultispecific antibody according to the invention, comprising the stepsof

-   -   transforming a host cell with expression vectors comprising        nucleic acids encoding the multispecific antibody,    -   culturing said host cell under conditions that allow synthesis        of said multispecific antibody, and    -   recovering said multispecific antibody from said host cell        culture.

In one embodiment of the invention, the method includes the step ofpurification of the multispecific antibody via affinity chromatography.In one embodiment of the invention, the method includes the step ofpurification of the multispecific antibody via affinity chromatographyon a kappa light chain or lambda light chain specific column.

Another object of the invention is a multispecific antibody produced bya method according to the invention.

Another object of the invention is a nucleic acid encoding themultispecific antibody according to the invention. In one embodiment,the nucleic acid according to the invention is an isolated nucleic acid.

Another object of the invention is an expression vector comprising anucleic acid according to the invention. Another object of the inventionis an expression vector comprising a nucleic acid according to theinvention, wherein the expression vector is capable of expressing saidnucleic acid in a host cell.

Another object of the invention is a host cell comprising a nucleic acidaccording to the invention. Another object of the invention is a hostcell comprising an expression vector according to the invention. In oneembodiment the host cell is a HEK293 cells or a CHO cell.

III. Pharmaceutical Composition

Another object of the invention is a pharmaceutical compositioncomprising a multispecific antibody according to the invention. Oneaspect of the invention is a pharmaceutical composition comprising amultispecific antibody according to the invention in combination with atleast one pharmaceutically acceptable carrier.

In one embodiment, a composition (in one preferred embodiment apharmaceutical composition) comprising a population of antibodies of theinvention comprises an antibody comprising a heavy chain including a CH3domain, as specified herein, with an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment, a composition comprising a population ofantibodies of the invention comprises an antibody comprising a heavychain including a CH3 domain, as specified herein, with an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat).

In one embodiment, such a composition comprises a population ofantibodies comprised of antibodies comprising a heavy chain including aCH3 domain, as specified herein; antibodies comprising a heavy chainincluding a CH3 domain, as specified herein, with an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat); and antibodies comprising a heavy chain including a CH3 domain,as specified herein, with an additional C-terminal glycine-lysinedipeptide (G446 and K447, numbering according to EU index of Kabat).

Another object of the invention is a pharmaceutical compositioncomprising a complex according to the invention comprising themultispecific antibody according to the invention, which specificallybinds to a hapten and a target protein, and a hapten that is conjugatedto a therapeutic or diagnostic agent. One aspect of the invention is apharmaceutical composition comprising a complex according to theinvention comprising the multispecific antibody according to theinvention, which specifically binds to a hapten and a target protein,and a hapten that is conjugated to a therapeutic or diagnostic agent incombination with at least one pharmaceutically acceptable carrier.

Another object of the invention is an immunoconjugate comprising themultispecific antibody according to the invention coupled to a cytotoxicagent.

Another object of the invention is a pharmaceutical compositioncomprising an immunoconjugate comprising the multispecific antibodyaccording to the invention coupled to a cytotoxic agent. One aspect ofthe invention is a pharmaceutical composition comprising animmunoconjugate comprising the multispecific antibody according to theinvention coupled to a cytotoxic agent in combination with at least onepharmaceutically acceptable carrier.

Another object of the invention is the use of a multispecific antibodyaccording to the invention for the manufacture of a pharmaceuticalcomposition. Another object of the invention is a method for themanufacture of a pharmaceutical composition comprising a multispecificantibody according to the invention, including formulating themultispecific antibody according to the invention in combination with atleast one pharmaceutically acceptable carrier.

Another object of the invention is the multispecific antibody accordingto the invention for use as a medicament. Another object of theinvention is the multispecific antibody according to the invention foruse in the treatment of cancer. Another object of the invention is themultispecific antibody according to the invention for use in thetreatment of inflammatory diseases, autoimmune diseases, rheumatoidarthritis, psoriatic arthritis, muscle diseases (e.g. musculardystrophy), multiple sclerosis, chronic kidney diseases, bone diseases(e.g. bone degeneration in multiple myeloma), systemic lupuserythematosus, lupus nephritis, and/or vascular injury.

Another object of the invention is a pharmaceutical compositioncomprising a multispecific antibody according to the invention incombination with at least one pharmaceutically acceptable carrier foruse as a medicament. Another object of the invention is a pharmaceuticalcomposition comprising a multispecific antibody according to theinvention in combination with at least one pharmaceutically acceptablecarrier for use in the treatment of cancer. Another object of theinvention is a pharmaceutical composition comprising a multispecificantibody according to the invention in combination with at least onepharmaceutically acceptable carrier for use in the treatment ofinflammatory diseases, autoimmune diseases, rheumatoid arthritis,psoriatic arthritis, muscle diseases (e.g. muscular dystrophy), multiplesclerosis, chronic kidney diseases, bone diseases (e.g. bonedegeneration in multiple myeloma), systemic lupus erythematosus, lupusnephritis, and/or vascular injury.

Another object of the invention is a complex according to the inventioncomprising the multispecific antibody according to the invention, whichspecifically binds to a hapten and a target protein, and a hapten thatis conjugated to a therapeutic or diagnostic agent for use as amedicament. Another object of the invention is a complex according tothe invention comprising the multispecific antibody according to theinvention, which specifically binds to a hapten and a target protein,and a hapten that is conjugated to a therapeutic or diagnostic agent foruse in the treatment of cancer. Another object of the invention is acomplex according to the invention comprising the multispecific antibodyaccording to the invention, which specifically binds to a hapten and atarget protein, and a hapten that is conjugated to a therapeutic ordiagnostic agent for use in the treatment of inflammatory diseases,autoimmune diseases, rheumatoid arthritis, psoriatic arthritis, musclediseases (e.g. muscular dystrophy), multiple sclerosis, chronic kidneydiseases, bone diseases (e.g. bone degeneration in multiple myeloma),systemic lupus erythematosus, lupus nephritis, and/or vascular injury.

Another object of the invention is an immunoconjugate comprising themultispecific antibody according to the invention coupled to a cytotoxicagent for use as a medicament. Another object of the invention is animmunoconjugate comprising the multispecific antibody according to theinvention coupled to a cytotoxic agent for use in the treatment ofcancer. Another object of the invention is an immunoconjugate comprisingthe multispecific antibody according to the invention coupled to acytotoxic agent for use in the treatment of inflammatory diseases,autoimmune diseases, rheumatoid arthritis, psoriatic arthritis, musclediseases (e.g. muscular dystrophy), multiple sclerosis, chronic kidneydiseases, bone diseases (e.g. bone degeneration in multiple myeloma),systemic lupus erythematosus, lupus nephritis, and/or vascular injury.

Another object of the invention is the use of a multispecific antibodyaccording to the invention for the manufacture of a medicament. Anotherobject of the invention is the use of a multispecific antibody accordingto the invention for the manufacture of a medicament for the treatmentof cancer. Another object of the invention is the use of a multispecificantibody according to the invention for the manufacture of a medicamentfor the treatment of inflammatory diseases, autoimmune diseases,rheumatoid arthritis, psoriatic arthritis, muscle diseases (e.g.muscular dystrophy), multiple sclerosis, chronic kidney diseases, bonediseases (e.g. bone degeneration in multiple myeloma), systemic lupuserythematosus, lupus nephritis, and/or vascular injury.

Another object of the invention is a method of treatment of a patientsuffering from a disease by administering a multispecific antibodyaccording to the invention to the patient in the need of such treatment.Another object of the invention is a method of treatment of a patientsuffering from cancer by administering a multispecific antibodyaccording to the invention to the patient in the need of such treatment.Another object of the invention is a method of treatment of a patientsuffering from at least one of the following diseases includinginflammatory diseases, autoimmune diseases, rheumatoid arthritis,psoriatic arthritis, muscle diseases (e.g. muscular dystrophy), multiplesclerosis, chronic kidney diseases, bone diseases (e.g. bonedegeneration in multiple myeloma), systemic lupus erythematosus, lupusnephritis, and vascular injury; by administering a multispecificantibody according to the invention to the patient in the need of suchtreatment.

3. Specific Embodiments of the Invention

In the following specific embodiments of the invention are listed.

-   1. A multispecific antibody comprising at least three antigen    binding sites, wherein two antigen binding sites are formed by a    first antigen binding moiety and a second antigen binding moiety,    wherein    -   a) a third antigen binding site is formed by a variable heavy        chain domain (VH₃) and a variable light chain domain (VL₃),        wherein        -   the N-terminus of the VH₃ domain is connected to the first            antigen binding moiety via a first peptide connector, and        -   the N-terminus of the VL₃ domain is connected to the second            antigen binding moiety via a second peptide connector,    -   b) the multispecific antibody comprises two constant heavy chain        domains 3 (CH3), which are altered to promote heterodimerization        by        -   i) generation of a protuberance in one of the CH3 domains by            substituting at least one original amino acid residue by an            amino acid residue having a larger side chain volume than            the original amino acid residue, and generation of a cavity            in the other one of the CH3 domains by substituting at least            one original amino acid residue by an amino acid residue            having a smaller side chain volume than the original amino            acid residue, such that the protuberance generated in one of            the CH3 domains is positionable in the cavity generated in            the other one of the CH3 domains; or        -    substituting at least one original amino acid residue in            one of the CH3 domains by a positively charged amino acid,            and substituting at least one original amino acid residue in            the other one of the CH3 domains by a negatively charged            amino acid;        -   ii) introduction of at least one cysteine residue in each            CH3 domain such that a disulfide bond is formed between the            CH3 domains, or        -   iii) both modifications of i) and ii);    -   c) the C-terminus of the VH₃ domain of the third antigen binding        site is connected to one of the CH3 domains, and the C-terminus        of the VL₃ domain of the third antigen binding site is connected        to the other one of the CH3 domains, and    -   d) the multispecific antibody is devoid of constant heavy chain        domains 2 (CH2).-   2. The multispecific antibody according to embodiment 1, wherein the    multispecific antibody comprises two constant heavy chain domains 3    (CH3), which are altered to promote heterodimerization by generation    of a protuberance in one of the CH3 domains by substituting at least    one original amino acid residue by an amino acid residue having a    larger side chain volume than the original amino acid residue, and    generation of a cavity in the other one of the CH3 domains by    substituting at least one original amino acid residue by an amino    acid residue having a smaller side chain volume than the original    amino acid residue, such that the protuberance generated in one of    the CH3 domains is positionable in the cavity generated in the other    one of the CH3 domains.-   3. The multispecific antibody according to embodiment 2, wherein    said amino acid residue having a larger side chain volume than the    original amino acid residue is selected from R, F, Y and W.-   4. The multispecific antibody according to embodiment 2 or 3,    wherein said amino acid residue having a smaller side chain volume    than the original amino acid residue is selected from A, S, T and V.-   5. The multispecific antibody according to any one of embodiments 2    to 4, wherein the CH3 domain of the one heavy chain comprises a    T366W mutation, and the CH3 domain of the other heavy chain (the    heavy chain comprising the “hole”) comprises T366S, L368A and 407V    mutations (numberings according to EU index of Kabat).-   6. The multispecific antibody according to any one of embodiments 2    to 5, wherein the CH3 domain of the one heavy chain comprises T366W    and G407Y mutations, and the CH3 domain of the other heavy chain    comprises T366S, L368A and Y407V mutations (numberings according to    EU index of Kabat).-   7. The multispecific antibody according to embodiment 1, wherein the    multispecific antibody comprises two constant heavy chain domains 3    (CH3), which are altered to promote heterodimerization by    substituting at least one original amino acid residue in one of the    CH3 domains by a positively charged amino acid, and substituting at    least one original amino acid residue in the other one of the CH3    domains by a negatively charged amino acid.-   8. The multispecific antibody according to any one of embodiments 2    to 6, wherein the multispecific antibody comprises two constant    heavy chain domains 3 (CH3), which are altered to promote    heterodimerization by substituting at least one original amino acid    residue in one of the CH3 domains by a positively charged amino    acid, and substituting at least one original amino acid residue in    the other one of the CH3 domains by a negatively charged amino acid.-   9. The multispecific antibody according to embodiment 7 or 8,    wherein said positively charged amino acid is selected from K, R and    H.-   10. The multispecific antibody according to any one of embodiments 7    to 9, wherein said negatively charged amino acid is selected from E    or D.-   11. The multispecific antibody according to any one of embodiments 7    to 10, wherein in the CH3 domain of one heavy chain the amino acid R    at position 409 (numbering according to EU index of Kabat) is    substituted by D and the amino acid K at position 370 (numbering    according to EU index of Kabat) is substituted by E; and in the CH3    domain of the other heavy chain the amino acid D at position 399    (numbering according to EU index of Kabat) is substituted by K and    the amino acid E at position 357 (numbering according to EU index of    Kabat) is substituted by K.-   12. The multispecific antibody according to embodiment 2, wherein    the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    introduction of at least one cysteine residue in each CH3 domain    such that a disulfide bond is formed between the CH3 domains.-   13. The multispecific antibody according to any one of embodiments 3    to 6, wherein the multispecific antibody comprises two constant    heavy chain domains 3 (CH3), which are altered to promote    heterodimerization by introduction of at least one cysteine residue    in each CH3 domain such that a disulfide bond is formed between the    CH3 domains.-   14. The multispecific antibody according to embodiment 7, wherein    the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    introduction of at least one cysteine residue in each CH3 domain    such that a disulfide bond is formed between the CH3 domains.-   15. The multispecific antibody according to embodiment 8, wherein    the multispecific antibody comprises two constant heavy chain    domains 3 (CH3), which are altered to promote heterodimerization by    introduction of at least one cysteine residue in each CH3 domain    such that a disulfide bond is formed between the CH3 domains.-   16. The multispecific antibody according to embodiment 9 or 10,    wherein the multispecific antibody comprises two constant heavy    chain domains 3 (CH3), which are altered to promote    heterodimerization by introduction of at least one cysteine residue    in each CH3 domain such that a disulfide bond is formed between the    CH3 domains.-   17. The multispecific antibody according to any one of embodiments    12 to 16, wherein the CH3 domains are disulfide stabilized by a    E356C or a S354C mutation in one of the CH3 domains and a Y349C    mutation in the other CH3 domain (numberings according to EU index    of Kabat.-   18. The multispecific antibody according to any one of embodiments    12 to 16, wherein the CH3 domains are disulfide stabilized by a    S354C mutation in one of the CH3 domains and a Y349C mutation in the    other CH3 domain (numberings according to EU index of Kabat).-   19. The multispecific antibody according to embodiment 1, wherein    the third binding site is disulfide stabilized by introduction of    cysteine residues at the following positions to form a disulfide    bond between the VH₃ and VL₃ domains (numbering according to Kabat):    -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.-   20. The multispecific antibody according to embodiment 2, wherein    the third binding site is disulfide stabilized by introduction of    cysteine residues at the following positions to form a disulfide    bond between the VH₃ and VL₃ domains (numbering according to Kabat):    -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.-   21. The multispecific antibody according to embodiment 7 or 8,    wherein the third binding site is disulfide stabilized by    introduction of cysteine residues at the following positions to form    a disulfide bond between the VH₃ and VL₃ domains (numbering    according to Kabat):    -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.-   22. The multispecific antibody according to any one of the preceding    embodiments, wherein the third binding site is disulfide stabilized    by introduction of cysteine residues at the following positions to    form a disulfide bond between the VH₃ and VL₃ domains (numbering    according to Kabat):    -   VH₃ at position 44, and VL₃ at position 100;    -   VH₃ at position 105, and VL₃ at position 43; or    -   VH₃ at position 101, and VL₃ at position 100.-   23. The multispecific antibody according to any one of embodiments    19 to 22, wherein the third binding site is disulfide stabilized by    introduction of cysteine residues in the VH₃ domain at position 44,    and in the VL₃ domain at position 100.-   24. The multispecific antibody according to any one of the preceding    embodiments, wherein no interchain disulfide bond is formed between    the first and the second peptide connector.-   25. The multispecific antibody according to any one of the preceding    embodiments, wherein the first and second peptide connectors are    identical to each other.-   26. The multispecific antibody according to any one of the preceding    embodiments, wherein the first and second peptide connector are    peptides of at least 15 amino acids.-   27. The multispecific antibody according to any one of the preceding    embodiments, wherein the first and second peptide connector are    peptides of 15-70 amino acids.-   28. The multispecific antibody according to any one of embodiments 1    to 25, wherein the first and second peptide connector are peptides    of 10-20 amino acids.-   29. The multispecific antibody according to any one embodiments 1 to    27, wherein the first and second peptide connector are peptides of    55-70 amino acids.-   30. The multispecific antibody according to any one of the preceding    embodiments, wherein the peptide connectors are glycine-serine    linkers.-   31. The multispecific antibody according to embodiment 30, wherein    the glycine-serine linkers are of the structure

(GxS)n or (GxS)nGm

-   -   with G=glycine, S=serine, x=3 or 4, n=2, 3, 4, 5 or 6, and m=0,        1, 2 or 3.

-   32. The multispecific antibody according to any one of the preceding    embodiments, wherein the variable domains VH₃ and VL₃ are directly    connected to the respective CH3 domains without the aid of a peptide    connector.

-   33. The multispecific antibody according to any one of the preceding    embodiments, wherein the C-terminus of the VH₃ domain is directly    connected to one of the CH3 domains, and the C-terminus of the VL₃    domain is directly connected to the other one of the CH3 domains,    wherein the connection sites are devoid of an additional linker    peptide.

-   34. The multispecific antibody according to any one of the preceding    embodiments, wherein the N-terminus of the CH3 domain is modified by    substituting at least one original amino acid residue.

-   35. The multispecific antibody according to embodiment 34, wherein    at least one amino acid residue located at positions 341 to 350 of    the CH3 domains (numbering according to EU index of Kabat) is    substituted.

-   36. The multispecific antibody according to embodiment 34 or 35,    wherein the N-terminus of the CH3 domain consists of an amino acid    sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

-   37. The multispecific antibody according to any one of the preceding    embodiments, wherein the C-terminus of the VH₃ domain is modified by    substituting at least one original amino acid residue.

-   38. The multispecific antibody according to any one of the preceding    embodiments, wherein the C-terminus of the VL₃ domain is modified by    substituting at least one original amino acid residue.

-   39. The multispecific antibody according to any one of the preceding    embodiments, wherein the N-terminus of each one of the CH3 domains    includes at least one amino acid mutation, the C-terminus of the VH₃    domain includes at least one amino acid mutation, and C-terminus of    the VL₃ domain includes at least one amino acid mutation.

-   40. The multispecific antibody according to any one of the preceding    embodiments, wherein the antigen binding moiety is a protein    specifically binding to an antigen.

-   41. The multispecific antibody according to embodiment 40, wherein    the antigen binding moiety is selected from the group of antibodies,    receptors, ligands, and DARPins capable of specifically binding to    an antigen.

-   42. The multispecific antibody according to embodiment 40, wherein    the antigen binding moiety is an antibody or an antibody fragment.

-   43. The multispecific antibody according to embodiment 42, wherein    at least one of the first and the second antigen binding moiety is a    Fab fragment.

-   44. The multispecific antibody according to embodiment 42, wherein    the first and the second antigen binding moiety are Fab fragments.

-   45. The multispecific antibody according to embodiment 44, wherein    the constant light chain domain of the first and/or the second Fab    fragment is of kappa isotype.

-   46. The multispecific antibody according to embodiment 44, wherein    the constant light chain domain of the first and/or the second Fab    fragment is of lambda isotype.

-   47. The multispecific antibody according to embodiment 44, wherein    the constant light chain domain of the first Fab fragment is of    kappa isotype and the constant light chain domain of the second Fab    fragment is of lambda isotype.

-   48. The multispecific antibody according to embodiment 43 or 44,    wherein at least one Fab fragment is disulfide-stabilized.

-   49. The multispecific antibody according to embodiment 44, wherein    the Fab fragments are disulfide-stabilized.

-   50. The multispecific antibody according to embodiment 43 or 44,    wherein the Fab fragments are disulfide stabilized by introduction    of cysteine residues at the following positions to form a disulfide    bond between the corresponding VH and VL domains (numbering    according to Kabat):    -   VH at position 44, and VL at position 100;    -   VH at position 105, and VL at position 43; or    -   VH at position 101, and VL at position 100.

-   51. The multispecific antibody according to any one of embodiments    48 to 50, wherein the natural disulfide bond between the polypeptide    chains of the respective Fab fragment is abolished by substituting    at least one of the interchain disulfide-bond-forming cysteine    residues by another amino acid residue.

-   52. The multispecific antibody according to embodiment 43, 44 or 48,    wherein at least one Fab fragment is a single chain Fab fragment.

-   53. The multispecific antibody according to embodiment 43, 44 or 48,    wherein at least one Fab fragment is altered by a domain crossover,    such that either:    -   a) only the CH1 and CL domains are replaced by each other;    -   b) only the VH and VL domains are replaced by each other; or    -   c) the CH1 and CL domains are replaced by each other and the VH        and VL domains are replaced by each other,    -   with the provision that in case both the first Fab fragment and        the second Fab fragment are altered by a domain crossover, they        are altered by different domain crossovers.

-   54. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody is trivalent.

-   55. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises at least one    polyepitopic binding site.

-   56. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody is trivalent.

-   57. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises three binding sites each    one binding to a single epitope.

-   58. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody is bispecific or trispecific.

-   59. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises at least one binding    site specifically binding to a hapten.

-   60. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises exactly one binding site    specifically binding to a hapten.

-   61. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises at least one binding    site specifically binding to a target protein.

-   62. The multispecific antibody according to embodiment 61, wherein    the target protein is a cell surface or an intracellular    tumor-associated antigen.

-   63. The multispecific antibody according to any one of the preceding    embodiments, wherein the antibody comprises at least one binding    site specifically binding to a hapten and at least one binding site    specifically binding to a target protein.

-   64. A complex comprising    -   (i) the multispecific antibody according to embodiment 63,        wherein the multispecific antibody specifically binds to a        hapten and a target protein, and    -   (ii) the hapten, which is specifically bound by the        multispecific antibody, wherein the hapten is conjugated to a        therapeutic or diagnostic agent.

-   65. The complex according to embodiment 64, wherein the hapten is    selected from digoxigenin, biotin, theophylline, fluorescein, DOTA,    and DOTAM.

-   66. The complex according to embodiment 64 or 65, wherein the target    protein is a cell surface antigen or an intracellular antigen.

-   67. The complex according to embodiment 66, wherein the target    protein is a cell surface or an intracellular tumor-associated    antigen.

-   68. The complex according to any one of embodiments 64 to 67,    wherein the therapeutic or diagnostic agent coupled to the hapten is    selected from the group consisting of a peptide, a protein, a small    molecule, a radioactively labeled small molecule, a nucleic acid and    an imaging agent.

-   69. A method for the preparation of the multispecific antibody    according to any one of embodiments 1 to 63, comprising the steps of    -   transforming a host cell with expression vectors comprising        nucleic acids encoding the multispecific antibody,    -   culturing said host cell under conditions that allow synthesis        of said multispecific antibody, and    -   recovering said multispecific antibody from said host cell        culture.

-   70. The method according to claim 69, further including the step of    purification of the multispecific antibody via affinity    chromatography on a kappa light chain or lambda light chain specific    column.

-   71. A multispecific antibody produced by the method according to    embodiment 69 or 70.

-   72. A method for the preparation of a complex, including the steps    of    -   providing a multispecific antibody according to embodiments 63,        wherein the multispecific antibody specifically binds to a        hapten and a target protein,    -   providing a hapten, which is specifically bound by the        multispecific antibody, wherein the hapten is conjugated to a        therapeutic or diagnostic agent, and    -   contacting the multispecific antibody with the hapten, which is        conjugated to the therapeutic or diagnostic agent.

-   73. A complex produced by the method according to embodiment 72.

-   74. A nucleic acid encoding the multispecific antibody according to    any one of embodiments 1 to 63.

-   75. An expression vector comprising a nucleic acid according to    embodiment 73.

-   76. A host cell comprising a nucleic acid according to embodiment    74.

-   77. A host cell comprising an expression vector according to    embodiment 75.

-   78. A composition comprising the multispecific antibody according to    any one of embodiments 1 to 63.

-   79. A pharmaceutical or diagnostic composition comprising the    multispecific antibody according to any one of embodiments 1 to 63.

-   80. A pharmaceutical composition comprising the multispecific    antibody according to any one of embodiments 1 to 63 in combination    with at least one pharmaceutically acceptable carrier.

-   81. A composition comprising the complex according to any one of    embodiments 64 to 68.

-   82. A pharmaceutical or diagnostic composition comprising the    complex according to any one of embodiments 64 to 68.

-   83. A pharmaceutical composition comprising the complex according to    any one of embodiments 64 to 68 in combination with at least one    pharmaceutically acceptable carrier.

-   84. A diagnostic composition comprising the complex according to any    one of embodiments 64 to 68.

-   85. An immunoconjugate comprising the multispecific antibody    according to any one of embodiments 1 to 63 coupled to a cytotoxic    agent.

-   86. The immunoconjugate according to embodiment 85, wherein the    multispecific antibody specifically binds to a cell surface    tumor-associated antigen or an intracellular tumor-associated    antigen.

-   87. A composition comprising the immunoconjugate according to    embodiment 85 or 86.

-   88. A pharmaceutical or diagnostic composition comprising the    immunoconjugate according to embodiment 85 or 86.

-   89. A pharmaceutical composition comprising the immunoconjugate    according to embodiment 85 or 86 in combination with at least one    pharmaceutically acceptable carrier.

-   90. A pharmaceutical composition comprising the multispecific    antibody according to embodiment 62, or a complex according to any    one of embodiments 64 to 68, or an immunoconjugate according to    embodiment 86, in combination with at least one pharmaceutically    acceptable carrier

-   91. The multispecific antibody according to any one of embodiments 1    to 63 for use as a medicament.

-   92. The multispecific antibody according to embodiment 62 for use as    a medicament.

-   93. The multispecific antibody according to embodiment 62 for use as    in the treatment of cancer.

-   94. The complex according to any one of embodiments 64 to 68 for use    as a medicament.

-   95. The complex according to any one of embodiments 64 to 68 for use    in the treatment of cancer.

-   96. The immunoconjugate according to embodiment 85 or 86 for use as    a medicament.

-   97. The immunoconjugate according to embodiment 86 for use as a    medicament.

-   98. The immunoconjugate according to embodiment 85 or 86 for the    treatment of cancer.

-   99. Use of the multispecific antibody according to any one of    embodiments 1 to 63 for diagnostic purposes.

-   100. Use according to embodiment 99 for imaging.

-   101. A method of treatment of a patient suffering from a disease by    administering a multispecific antibody according to any one of    embodiments 1 to 63 to the patient in the need of such treatment.

-   102. A method of treatment of a patient suffering from a disease by    administering a multispecific antibody according to any one of    embodiments 62 or 63 to the patient in the need of such treatment.

-   103. The method according to embodiment 102, wherein the disease is    cancer.

-   104. A method of treatment of a patient suffering from a disease by    administering a complex according to any one of embodiments 64 to 68    to the patient in the need of such treatment.

-   105. The method according to embodiment 104, wherein the disease is    cancer.

-   106. A method of treatment of a patient suffering from a disease by    administering an immunoconjugate according to embodiment 85 or 86 to    the patient in the need of such treatment.

-   107. A method of treatment of a patient suffering from a disease by    administering an immunoconjugate according to embodiment 86 to the    patient in the need of such treatment.

-   108. The method according to embodiment 106 or 107, wherein the    disease is cancer.

-   109. The method according to any one of embodiments 101, 104 and    106, wherein the disease is selected from inflammatory diseases,    autoimmune diseases, rheumatoid arthritis, psoriatic arthritis,    muscle diseases, multiple sclerosis, chronic kidney diseases, bone    diseases, systemic lupus erythematosus, lupus nephritis, and/or    vascular injury.

Description of the Amino Acid Sequences

SEQ ID NO: 1 exemplary fusion site of <anti-DIG> VL₃—CH3 fusion site ofan antibody according to example 1 SEQ ID NO: 2 N-terminus of CH3domains (alternative 1) SEQ ID NO: 3 N-terminus of CH3 domains(alternative 2) SEQ ID NO: 4 N-terminus of CH3 domains (alternative 3)SEQ ID NO: 5 light chain polypeptide with digoxigenin binding site SEQID NO: 6 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb Dig-LeY-Dig SEQ IDNO: 7 polypeptide VH—CH1-linker-VL₃—CH3 of BsAb Dig-LeY-Dig SEQ ID NO: 8light chain polypeptide with Lewis Y binding site SEQ ID NO: 9polypeptide VH—CH1-linker-VH₃—CH3 of BsAb LeY-Dig(SS)-LeY SEQ ID NO: 10polypeptide VH—CH1-linker-VL₃—CH3 of BsAb LeY-Dig(SS)-LeY SEQ ID NO: 11polypeptide VH—CH1-linker-VH₃—CH3 of BsAb Dig-CD33-Dig SEQ ID NO: 12polypeptide VH—CH1-linker-VL₃—CH3 of BsAb Dig-CD33-Dig SEQ ID NO: 13light chain polypeptide with CD33 binding site SEQ ID NO: 14 polypeptideVH—CH1-linker-VH₃—CH3 of BsAb CD33- Dig(SS)-CD33 SEQ ID NO: 15polypeptide VH—CH1-linker-VL₃—CH3 of BsAb CD33- Dig(SS)-CD33 SEQ ID NO:16 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb Dig-GPC3-Dig SEQ ID NO: 17polypeptide VH—CH1-linker-VL₃—CH3 of BsAb Dig-GPC3-Dig SEQ ID NO: 18light chain polypeptide with Glypican 3 binding site SEQ ID NO: 19polypeptide VH—CH1-linker-VH₃—CH3 of BsAb GPC3- Dig(SS)-GPC3 SEQ ID NO:20 polypeptide VH—CH1-linker-VL₃—CH3 of BsAb GPC3- Dig(SS)-GPC3 SEQ IDNO: 21 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb LeY-Bio(SS)-LeY SEQ IDNO: 22 polypeptide VH—CH1-linker-VL₃—CH3 of BsAb LeY-Bio(SS)-LeY SEQ IDNO: 23 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb CD33- Bio(SS)-CD33 SEQID NO: 24 polypeptide VH—CH1-linker-VL₃—CH3 of BsAb CD33-Bio(SS)-CD33SEQ ID NO: 25 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb GPC3-Bio(SS)-GPC3 SEQ ID NO: 26 polypeptide VH—CH1-linker-VL₃—CH3 of BsAbGPC3- Bio(SS)-GPC3 SEQ ID NO: 27 light chain polypeptide with Biotinbinding site SEQ ID NO: 28 polypeptide VH—CH1-linker-VH₃—CH3 of BsAbBio-LeY(SS)-Bio SEQ ID NO: 29 polypeptide VH—CH1-linker-VL₃—CH3 of BsAbBio-LeY(SS)-Bio SEQ ID NO: 30 polypeptide VH—CH1-linker-VH₃—CH3 of BsAbBio- CD33(SS)-Bio SEQ ID NO: 31 polypeptide VH—CH1-linker-VL₃—CH3 ofBsAb Bio-CD33(SS)-Bio SEQ ID NO: 32 polypeptide VH—CH1-linker-VH₃—CH3 ofBsAb Bio- GPC3(SS)-Bio SEQ ID NO: 33 polypeptide VH—CH1-linker-VL₃—CH3of BsAb Bio- GPC3(SS)-Bio SEQ ID NO: 34 polypeptideVH—CH1-linker-VH₃—CH3 of BsAb Dig-LeY(SS)-Dig SEQ ID NO: 35 polypeptideVH—CH1-linker-VL₃—CH3 of BsAb Dig-LeY(SS)-Dig SEQ ID NO: 36 polypeptideVH—CH1-linker-VH₃—CH3 of BsAb Dig- CD33(SS)-Dig SEQ ID NO: 37polypeptide VH—CH1-linker-VL₃—CH3 of BsAb Dig- CD33(SS)-Dig SEQ ID NO:38 polypeptide VH—CH1-linker-VH₃—CH3 of BsAb Dig- GPC3(SS)-Dig SEQ IDNO: 39 polypeptide VH—CH1-linker-VL₃—CH3 of BsAb Dig- GPC3(SS)-Dig

EXAMPLES

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1

Production and Expression of Trivalent, Bispecific Antibodies Accordingto the Invention Specifically Binding to Digoxigenin (Dig) and One ofthe Cell Surface Antigens Lewis-Y (LeY), CD33 and Glypican3 (GPC3)

Bispecific antibodies comprising three antigen binding sites weredesigned in an IgG-like structure being composed of two regular Fab armsas first and second binding moieties, which were fused via flexibleglycine-serine peptide linkers to a third approximately Fab-sizedbinding module. This third binding module replaces the original IgG Fcregion of a full length antibody and is composed of a variable heavychain domain VH₃ fused to a first CH3 domain, and a variable light chaindomain VL₃ fused to a second CH3 domain (FIG. 2A). The domainarchitecture of the bispecific antibodies is indicated in FIGS. 2A and2B (illustrating antibodies with and without an additional disulfidebond present within the third binding site).

The amino acid sequences of the third binding module, in particular thefusion site of the CH3 domains with either VH₃ or VL₃ were designed in amanner that possesses no strain or sterical disturbance on the overallIgG like structure, and retains IgG like properties.

The Fab fragments are fused to third binding module via peptideconnectors that are designed to replace the original hinge region of afull length IgG (FIGS. 4A and 4B). The peptide connectors do not containinterchain disulfide bridges, which facilitates antigen access to thethird binding site. Loss of hinge disulfide bridges however alsodestabilizes the antibody derivative as it removes the covalentinterchain connection. To compensate the loss of hinge-disulfides, andto regain stability, heterodimerization strategies to promoteheterodimerization of the domains of the third binding module wereapplied (see FIG. 2A).

The bispecific antibody molecules generated in this example comprisedisulfide-stabilized CH3-domains with either a “knob” (protuberance) ora “hole” (cavity) modification (FIGS. 2A and 2B). Within this examples,antibodies with and without disulfide-stabilization in the VH₃ and VL₃domains were provided (see Table 2).

The bispecific antibodies according to Table 1 were generated byclassical molecular biology techniques and expressed transiently inHEK293 suspension cells.

Briefly, the antibodies were produced by co-transfection of expressionvectors that encode the light chains of the desired antibodies withexpression vectors encoding the two corresponding “heavy chains” (i.e.the polypeptides of the domain structures VH-CH1-linker-VH₃-CH3 andVH-CH1-linker-VL3-CH3, respectively). The expression cassettes, plasmidproperties and conditions for transient expression were the same asdescribed by Metz et al. Protein Engineering Design and SelectionSeptember 2012; 25(10):571-8 and in WO 2012025525 A1, both documents areherein included by reference. The polypeptide components of theantibodies were expressed by CMV promoter driven transcription in HEK293suspension cells that were grown at 37° C. in a humidified 8% CO₂environment. 7 days after transfection, culture supernatants thatcontained the secreted bispecific antibodies were sterile filtered.

TABLE 1 Domain architecture of indicated bispecific antibodies Fab3^(rd) binding fragments site derived molecule name derived from fromBsAb Dig-LeY-Dig <Dig> <LeY> BsAb LeY-Dig(SS)-LeY <LeY> <Dig> BsAbDig-CD33-Dig <Dig> <CD33> BsAb CD33-Dig(SS)-CD33 <CD33> <Dig> BsAbDig-GPC3-Dig <Dig> <GPC3> BsAb GPC3-Dig(SS)-GPC3 <GPC3> <Dig>

The bispecific antibodies included the characteristics indicated inTable 2. All constructs comprised constant light chain domains of kappaisotype. In addition, in all constructs, the “knobs” substitutions wereintroduced in the CH3 domain fused to VH₃ and the “hole” substitutionswere introduced in the CH3 domain fused to VL₃. However with the sameeffect, the “knob” may be introduced into the CH3 domain fused to VL₃and the “hole” may be introduced into the CH3 domain fused to VH₃.

TABLE 2 Characteristics of indicated bispecific antibodies S—S bondknobs-into-holes S—S 1^(st) and 2^(nd) between substitutions in betweenpeptide VH₃ and CH3/CH3 CH3 and molecule name connector VL₃ interfaceCH3 BsAb Dig- (Gly₄Ser)₄ — Trp366, Tyr407 Cys354 LeY-Dig (knob); (knob);Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb LeY- (Gly₄Ser)₄ VH₃Cys44 Trp366, Tyr407 Cys354 Dig(SS)-LeY VL₃ Cys100 (knob); (knob);Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb Dig- (Gly₄Ser)₄ —Trp366, Tyr407 Cys354 CD33-Dig (knob); (knob); Ser366, Ala368, Cys349Val407 (hole) (hole) BsAb CD33- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407Cys354 Dig(SS)-CD33 VL₃ Cys100 (knob); (knob); Ser366, Ala368, Cys349Val407 (hole) (hole) BsAb Dig- (Gly₄Ser)₄ — Trp366, Tyr407 Cys354GPC3-Dig (knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole)BsAb GPC3- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 Dig(SS)-GPC3 VL₃Cys100 (knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole)

The amino acid sequences of the polypeptide chains of the testedbispecific antibodies are indicated in Table 3.

TABLE 3 Amino acid sequences of polypeptide chains of indicatedbispecific antibodies polypeptide polypeptide light VH—CH1- VH—CH1-chains linker- linker- SEQ VH₃—CH3 VL₃—CH3 molecule name ID NO: SEQ IDNO: SEQ ID NO: BsAb Dig-LeY-Dig 5 6 7 BsAb LeY-Dig(SS)-LeY 8 9 10 BsAbDig-CD33-Dig 5 11 12 BsAb CD33-Dig(SS)-CD33 13 14 15 BsAb Dig-GPC3-Dig 516 17 BsAb GPC3-Dig(SS)-GPC3 18 19 20

Example 2

Purification and Characterization of Trivalent, Bispecific AntibodiesAccording to the Invention

The bispecific antibodies expressed above in example 1 were purifiedfrom the supernatant by affinity chromatography via a HiTrap KappaSelectcolumn (GE Healthcare), as due to the lack of CH2 domains the bispecificantibodies do not bind to protein A. In a second purification step,homogeneous bispecific antibodies were obtained by applying sizeexclusion chromatography (SEC, Superdex200 HiLoad 26/60, GE Healthcare)equilibrated with 20 mM histidin, 140 mM NaCl, at pH 6.0 on an AektaAvant (GE Healthcare) as previously described for IgG-derived bispecificantibodies (S. Metz et al., Proc. Natl. Acad. Sci. U.S.A. 108 (2011)8194-8199).

Exemplary results of these purification steps for bispecific antibodieswith different specificities as indicated in Table 1 are shown in FIGS.6A and 6B. SDS PAGE analyses that demonstrate identity and purity of thegenerated multispecific antibodies are also shown in FIGS. 6A and 6B.

The bispecific antibodies could be generated by the described productionand purification method with yields between 3-20 mg/L, as indicated indetail in Table 4.

TABLE 4 Yield of indicated bispecific antibodies molecule name Yield[mg/L] BsAb Dig-LeY-Dig 5.7 BsAb LeY-Dig(SS)-LeY 3.0 BsAb Dig-CD33-Dig6.9 BsAb CD33-Dig(SS)-CD33 20.3 BsAb Dig-GPC3-Dig 8.3 BsAbGPC3-Dig(SS)-GPC3 3.5

Example 3

Design of Complex Disulfide Pattern to Promote Heterodimerization

Within the bispecific antibodies generated in example 1,heterodimerization of the third binding module is promoted by fourdistinct interactions: (i) the interaction between VH₃ and VL₃, (ii) thedisulfide stabilization in the VH₃/VL₃ interface, (iii) the disulfidestabilization in the CH3/CH3 interface; and (iv) the knobs-into-holesmodifications in the CH3/CH3 interface (which may be alternativelyreplaced by other comparable heterodimerization strategies known tosupport interaction of CH3 domains, like e.g. the introduction ofoppositely charged amino acids within the CH3/CH3 interface). By this,formation of heterodimers rather than homodimer formation is promoted.

As the disulfide stabilizations of the VH₃/VL₃ as well as the CH3/CH3interface required introduction of additional disulfides in closeproximity, it was necessary to avoid the formation of mispaireddisulfides during the production process leading to misfolded andnon-functional molecules (FIG. 5A).

As it is well-known, wild type full length IgG possess one intrachaindisulfide bond within each of its domains as well as interchaindisulfide bonds to connect the heavy chains via the hinge regions of theantibody. The hinge region cysteines do not interfere with folding ofthe individual antibody domains and do not interact in intradomaindisulfide formation.

However, within antibodies according to the invention comprisingdisulfide stabilizations within the VH₃/VL₃ interface as well as in theCH3/CH3 interface, additional unpaired cysteines are introduced, whichin order to assure correct folding must not pair with intrachaindisulfide bond forming cysteines but instead must form defined separateinterdomain bonds. Within the antibodies according to the invention, thevariable domains are directly connected to their respective CH3 domains(i.e. by peptide bond formation between amino acids of a variable domainwith an amino acid of a CH3 domain, without including a peptide linker).It was expected that such a sequence composition and close proximity ofcysteine residues (including the natural and the additionally introducedcysteine residues) would favor to a large degree the formation ofmispaired disulfides (FIG. 5A).

Therefore, a fusion site between the variable domains (VH₃ and VL₃) andtheir respective CH3 domains was created (FIG. 5B), which allows correctdisulfide bond formation and avoids disulfide mispairing. In eachpolypeptide chain of the third binding module, the cysteine residuesnecessary for the additional disulfide stabilization are in closeproximity to cysteine residues required for intrachain disulfide bondformation, but nevertheless surprisingly do not interfere with theintrachain disulfides and pair with the correct corresponding cysteineresidue in the other polypeptide chain.

FIG. 5B depict fusion sites including such a complex cysteine pattern inthe third binding module, which allow correct protein folding.

Another goal of the design of the fusion site between the variabledomains and the CH3 domains was to closely mimic the natural transitionsites present in the original parent antibody between (i) the VH and CH1domains as well as the CH2 and CH3 domains for the fusion site betweenVH₃ and CH3; and (ii) the VL and CL domains as well as the CH2 and CH3domains for the fusion site between VL₃ and CH3. Therefore, distinctamino acid residues at the C-terminus of the variable domains and theN-terminus of the CH3 domains may be substituted by another amino acidresidue in order to provide a fusion site of a tertiary structure of adistinct homology to the natural transition site between variable andconstant regions. Exemplary alternative amino acid sequences of theN-terminus of the CH3 domains are indicated in FIG. 5B.

Example 4

Binding Studies of Trivalent, Bispecific Antibodies According to theInvention

Simultaneous antigen binding of the antibodies generated in example 1was analyzed by FACS analysis on LeY-expressing MCF7 cells,CD33-expressing Molm13 cells and GPC3-expressing HepG2 cells usingDig-Cy5 as payload to address hapten binding. Results are shown in FIG.7.

Simultaneous hapten binding and cell surface binding was observed forall bispecific antibodies with the Dig-specific binding site in thethird binding module and the antigen binding sites specific for therespective cell surface marker within the Fab fragments. Simultaneoushapten binding and cell surface binding was also observed for theantibodies with the Dig-specific binding sites in the Fab fragments andthe antigen binding sites specific for the respective cell surfacemarker in the third binding module.

The data indicate that the third binding site is easily accessible forsmall antigens, e.g. haptens (like digoxigenin). The third binding siteis also accessible for binding larger antigens such as proteins, e.g.cell surface proteins. For those antigens, binding efficacy may dependon epitope accessibility and potential steric hindrance. Asdemonstrated, cell surface antigens CD33, GPC3 or LeY are accessible tothe third binding site of an antibody according to the invention.

Due to these characteristics, the antibodies according to the inventionmay can be applied to simultaneously address or crosslink two targets,e.g. for imaging, or for targeted payload delivery.

Example 5

Application of Trivalent, Bispecific Antibodies According to theInvention for Targeted Payload Delivery

Antibodies according to the invention (BsAb LeY-Dig(SS)-LeY) provided inexample 1 and 2 were complexed with a digoxigeninylated truncatedPseudomonas Exotoxin (PE) derivative as toxic payload. A schematicillustration of such complex is indicated in FIG. 9. In order to assesswhether the complexes including the antibodies according to theinvention are capable of targeting cancer cells thereby inducing celldeath, cells of a LeY-expressing MCF7 breast cancer cell line werecontacted in vitro with the complexes. Induction of cell death wasassessed by a commercial BrdU assay (Cell Proliferation ELISA, BrdU(chemoluminescence), Cat. No. 11 669 915 001, Roche) according to themanufacturer's instructions.

The following controls were analysed in parallel:

-   -   staurosporin (positive control),    -   no additives (medium only, negative control)    -   free PE,    -   free digoxigeninylated PE (PE-Dig),    -   free antibody BsAb LeY-Dig(SS)-LeY that was not complexed with        PE-Dig,    -   free antibody BsAb GPC3-Dig(SS)-GPC3 (which does not bind to        MCF7 breast cancer cells),    -   complex of BsAb CD33-Dig(SS)-CD33 (provided in examples 1 and 2)        with digoxigeninylated PE (negative control to assess the        unspecific activity of a complex including an antibody according        to the invention and a toxin as CD33 does not bind to MCF7        breast cancer cells; demonstrated in FIG. 7).

The following comparative example was analysed in parallel:

As complexes of bispecific antibodies specifically binding todigoxigenin and a target antigen with digoxigenin-coupled smallmolecules are known in the art (disclosed in WO 2011/1003557 A1, domainarchitecture as indicated in FIG. 17a ), such antibody complex was usedas a comparative example. In brief, the bispecific antibody was composedof a full length antibody specifically binding to LeY with an Fvfragment specifically binding to Dig fused to the C-terminus of eachheavy chain (referred to as “BsAb LeY-Dig(2+2)”). Variable domains ofthis antibody were the same as the BsAb LeY-Dig(SS)-LeY antibodyaccording to the invention.

The results (FIG. 10) demonstrate that only the complex of the BsAbLeY-Dig(SS)-LeY antibody according to the invention and thedigoxigeninylated PE are capable of effectively targeting MCF7 breastcancer cells in low nanomolar concentrations indicating specifictargeting of the cancer cells. To the contrary, complexes that addressthe CD33 antigen which is not present on MCF7 breast cancer cells showbarely any toxicity. In addition, toxin without targeting vehicles orthe control-antibodies without a toxic payload did not show noticeableactivity.

Example 6

Production and Analysis of Trivalent, Bispecific Antibodies According tothe Invention Specifically Binding to Biotin (Bio) and One of the CellSurface Antigens Lewis-Y (LeY), CD33 and Glypican3 (GPC3)

Bispecific antibodies comprising three antigen binding sites with thesame domain architecture specifically binding to biotin and LeY, orbiotin and CD33, or biotin and GPC3 were designed as described for theantibodies according to example 1. The domain architecture of thebispecific antibodies is indicated in FIGS. 2A and 2B indicating thatFab fragments were used as first and second antigen binding site.

TABLE 5 Domain architecture of indicated bispecific antibodies Fabfragments 3^(rd) binding site molecule name derived from derived fromBsAb LeY-Bio(SS)-LeY <LeY> <Bio> BsAb CD33-Bio(SS)-CD33 <CD33> <Bio>BsAb GPC3-Bio(SS)-GPC3 <GPC3> <Bio>

The bispecific antibodies included the characteristics indicated inTables 5 and 6. All constructs comprised constant light chain domains ofkappa isotype. In addition, in all constructs, the “knobs” substitutionswere introduced in the CH3 domain fused to VH₃ and the “hole”substitutions were introduced in the CH3 domain fused to VL₃.

TABLE 6 Characteristics of indicated bispecific antibodies S—S bondknobs-into-holes S—S 1^(st) and 2^(nd) between substitutions in betweenpeptide VH3 and CH3/CH3 CH3 and molecule name connector VL3 interfaceCH3 BsAb LeY- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 Bio(SS)-LeY VL₃Cys100 (knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAbCD33- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 Bio(SS)-CD33 VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb GPC3-(Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 Bio(SS)-GPC3 VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole)

The amino acid sequences of the polypeptide chains of the testedbispecific antibodies are indicated in Table 7.

TABLE 7 Amino acid sequences of polypeptide chains of indicatedbispecific antibodies polypeptide polypeptide light VH—CH1- VH—CH1-chains linker- linker- SEQ VH₃—CH3 VL₃—CH3 molecule name ID NO: SEQ IDNO: SEQ ID NO: BsAb LeY-Bio(SS)-LeY 8 21 22 BsAb CD33-Bio(SS)-CD33 13 2324 BsAb GPC3-Bio(SS)-GPC3 18 25 26

The three antibodies were transiently expressed in HEK293 cells andpurified as described in examples 1 and 2.

TABLE 8 Yield of indicated bispecific antibodies molecule name Yield[mg/L] BsAb LeY-Bio(SS)-LeY 3.8 BsAb CD33-Bio(SS)-CD33 8.3 BsAbGPC3-Bio(SS)-GPC3 3.8

In order to test simultaneous antigen binding of the BsAbLeY-Bio(SS)-LeY, the antibody was analyzed by FACS analysis onLeY-expressing MCF7 cells using biotinylated Cy5 as payload. The resultsof the FACS analysis are shown in FIG. 8.

Simultaneous binding to biotin and the MCF7 cell surface was observedfor the BsAb LeY-Bio(SS)-LeY antibody. As a control, a complex of theBsAb CD33-Bio(SS)-CD33 with biotinylated Cy5 as payload was run inparallel, demonstrating that no unspecific binding of the complex tocancer cells was identified. The results that the third binding site ofthe antibody according to the invention is easily accessible for haptenbinding, specifically for binding to biotin and biotin-coupled payloads.

Example 7

Application of Trivalent, Bispecific Antibodies According to theInvention for Targeted Payload Delivery

Using the same approach as described in example 5, the antibodiesaccording to the invention prepared in example 6 were assessed for theirapplicability in targeted payload delivery. For this, the antibodieswere complexed with a biotinylated truncated Pseudomonas Exotoxin (PE)derivative as toxic payload. A schematic illustration of such complex isindicated in FIG. 9.

In order to assess whether the complexes of BsAb LeY-Bio(SS)-LeY withbiotinylated PC are capable of targeting cancer cells thereby inducingcell death, the cells of a LeY-expressing MCF7 breast cancer cell linewere contacted in vitro with the complexes. Induction of cell death wasassessed by a commercial BrdU assay (Cell Proliferation ELISA, BrdU(chemoluminescence), Cat. No. 11 669 915 001, Roche) according to themanufacturer's instructions.

The following controls were analysed in parallel:

-   -   staurosporin (positive control),    -   no additives (medium only, negative control)    -   free biotinylated PE (PE-Bio),    -   complex of BsAb CD33-Bio(SS)-CD33 (provided in examples 1 and 2)        with biotinylated PE (negative control to assess the unspecific        activity of a complex including an antibody according to the        invention and a toxin as CD33 does not bind to MCF7 breast        cancer cells; demonstrated in FIG. 7).

The results (FIG. 11) demonstrate that only the complex of the BsAbLeY-Bio(SS)-LeY antibody according to the invention and the biotinylatedPE are capable of effectively targeting MCF7 breast cancer cells in lownanomolar concentrations indicating specific targeting of the cancercells. To the contrary, complexes that address the CD33 antigen which isnot present on MCF7 breast cancer cells show barely any toxicity. Inaddition, toxin without targeting vehicles or the control-antibodieswithout a toxic payload did not show noticeable activity.

Example 8

Production Trivalent, Bispecific Antibodies According to the InventionSpecifically Binding to Either Biotin (Bio) or Digoxigenin (Dig) inCombination with One of the Cell Surface Antigens Lewis-Y (LeY), CD33and GPC3

Bispecific antibodies comprising three antigen binding sites with thesame domain architecture specifically binding to either biotin (Bio) ordigoxigenin (Dig) in combination with one of the cell surface antigensLewis-Y (LeY), CD33 and GPC3 were designed as described for theantibodies according to example 1. The domain architecture of thebispecific antibodies is indicated in FIGS. 2A and 2B indicating thatFab fragments are used as first and second antigen binding site.

The bispecific antibodies include the characteristics indicated inTables 9 and 10. All constructs comprise constant light chain domains ofkappa isotype. In addition, in all constructs, the “knobs” substitutionsare introduced in the CH3 domain fused to VH₃ and the “hole”substitutions are introduced in the CH3 domain fused to VL₃.

TABLE 9 Domain architecture of indicated bispecific antibodies Fabfragments 3^(rd) binding derived site derived molecule name from fromBsAb Bio-LeY(SS)-Bio <Bio> <LeY> BsAb Bio-CD33(SS)-Bio <Bio> <CD33> BsAbBio-GPC3(SS)-Bio <Bio> <GPC3> BsAb Dig-LeY(SS)-Dig <Dig> <LeY> BsAbDig-CD33(SS)-Dig <Dig> <CD33> BsAb Dig-GPC3(SS)-Dig <Dig> <GPC3>

TABLE 10 Characteristics of indicated bispecific antibodies S—S bondknobs-into-holes S—S 1^(st) and 2^(nd) between substitutions in betweenpeptide VH₃ and CH3/CH3 CH3 and molecule name connector VL₃ interfaceCH3 BsAb Bio- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 LeY(SS)-Bio VL₃Cys100 (knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAbBio- (Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 CD33(SS)-Bio VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb Bio-(Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 GPC3(SS)-Bio VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb Dig-(Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 LeY(SS)-Dig VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb Dig-(Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 CD33(SS)-Dig VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole) BsAb Dig-(Gly₄Ser)₄ VH₃ Cys44 Trp366, Tyr407 Cys354 GPC3(SS)-Dig VL₃ Cys100(knob); (knob); Ser366, Ala368, Cys349 Val407 (hole) (hole)

The amino acid sequences of the polypeptide chains of the testedbispecific antibodies are indicated in Table 11.

TABLE 11 Amino acid sequences of polypeptide chains of indicatedbispecific antibodies light polypeptide polypeptide chainsVH—CH1-linker- VH—CH1-linker- SEQ ID VH₃—CH3 VL₃—CH3 molecule name NO:SEQ ID NO: SEQ ID NO: BsAb Bio-LeY(SS)-Bio 27 28 29 BsAbBio-CD33(SS)-Bio 27 30 31 BsAb Bio-GPC3(SS)-Bio 27 32 33 BsAbDig-LeY(SS)-Dig 5 34 35 BsAb Dig-CD33(SS)-Dig 5 36 37 BsAbDig-GPC3(SS)-Dig 5 38 39

The antibodies are expressed and purified as described in examples 1 and2.

1: A multispecific antibody comprising at least three antigen binding sites, wherein two antigen binding sites are formed by a first Fab fragment comprising a constant heavy chain domain (CH1) and a constant light chain domain (CL) and a second Fab fragment comprising a constant heavy chain domain (CH1) and a constant light chain domain (CL), wherein a) a third antigen binding site is formed by a variable heavy chain domain (VH₃) and a variable light chain domain (VL₃), wherein the N-terminus of the VH₃ domain is connected to the C-terminus of the CH1 or CL of the first Fab fragment via a first peptide connector, and the N-terminus of the VL₃ domain is connected to the C-terminus of the CH1 or CL of the second Fab fragment via a second peptide connector, wherein the third binding site is disulfide stabilized by introduction of cysteine residues at the following positions to form a disulfide bond between the VH₃ and VL₃ domains (numbering according to Kabat): (i) VH₃ at position 44, and VL₃ at position 100; (ii) VH₃ at position 105, and VL₃ at position 43; or (iii) VH₃ at position 101, and VL₃ at position 100; b) the multispecific antibody comprises a first and a second constant heavy chain domains 3 (CH₃), which are altered to promote heterodimerization by i) generation of a protuberance in the first CH₃ domain by substituting at least one original amino acid residue by an amino acid residue having a larger side chain volume than the original amino acid residue, and generation of a cavity in the second CH₃ domain by substituting at least one original amino acid residue by an amino acid residue having a smaller side chain volume than the original amino acid residue, such that the protuberance generated in the first CH₃ domain is positionable in the cavity generated in the second CH₃ domain; or substituting at least one original amino acid residue in the first CH₃ domain by a positively charged amino acid, and substituting at least one original amino acid residue in the second CH₃ domain by a negatively charged amino acid; ii) introduction of at least one cysteine residue in each CH₃ domain such that a disulfide bond is formed between the CH₃ domains, or iii) both modifications of i) and ii); c) the C-terminus of the VH₃ domain of the third antigen binding site is connected to one of the CH₃ domains, and the C-terminus of the VL₃ domain of the third antigen binding site is connected to the other one of the CH₃ domains, and d) the multispecific antibody is devoid of constant heavy chain domains 2 (CH₂). 2-3. (canceled) 4: The multispecific antibody according to claim 1, wherein the first and second peptide connector are peptides of at least 15 amino acids. 5: The multispecific antibody according to claim 1, wherein no interchain disulfide bond is formed between the first and the second peptide connector. 6: The multispecific antibody according to claim 1, wherein the C-terminus of the VH₃ domain is directly connected to one of the CH₃ domains, and the C-terminus of the VL₃ domain is directly connected to the other one of the CH₃ domains. 7: The multispecific antibody according to claim 1, wherein the antibody is trivalent. 8-13. (canceled) 14: A pharmaceutical composition comprising the multispecific antibody according to claim 8, in combination with at least one pharmaceutically acceptable carrier. 15: An immunoconjugate comprising the multispecific antibody according to claim 1 coupled to a cytotoxic agent. 16: The multispecific antibody according to claim 1, wherein the antibody is bispecific. 